Effect of Cholecalciferol on Mucin-1 Levels in Urinary Extracelllular Vesicles in Individuals With Autosomal Dominant Tubulo-Interstitial Kidney Disease Due to MUC1 Mutations (ADTKD-MUC1)

1. ADTKD-MUC1: Clinical Characteristics

   Autosomal dominant tubulointerstitial kidney disease due to MUC1 mutations (ADTKD-MUC1) is caused by mutations in the MUC1 gene, encoding the protein mucin-1. The most frequently reported mutation is a cytosine insertion causing a frameshift and resulting in the production of an abnormal protein (MUC1-fs). The primary characteristic of ADTKD-MUC1 is slowly progressive chronic kidney disease. Disease progression varies considerably, with affected individuals reaching end-stage kidney disease (ESRD), requiring dialysis or kidney transplantation, between 21 and over 70 years of age, with a median age of approximately 45 years. The reasons underlying variation in disease progression remain unclear.

   Although mucin-1 is expressed in numerous epithelial tissues, including sebaceous glands, the respiratory tract, mammary glands, and gastrointestinal tract, ADTKD-MUC1 is not typically associated with clinical manifestations outside the kidney. The Pafos region of Cyprus is home to one of the largest reported cohorts of patients with ADTKD-MUC1 worldwide, with an estimated prevalence of the MUC1 cytosine insertion of approximately 1 in 580 individuals. The chromosomal region at 1q22 associated with the disease was initially identified by a research group in Cyprus. The Cypriot ADTKD-MUC1 cohort has subsequently been extensively characterized and followed longitudinally.

2. Mucin-1

   Mucin-1 is a type I transmembrane heterodimer composed of two subunits. The larger alpha subunit is extracellular and consists of an N-terminal region of 104 amino acids, a variable number of tandem repeats (VNTRs), each comprising 20 amino acids and repeated approximately 25 to 125 times, and a C-terminal region of 170 amino acids. The alpha subunit is non-covalently associated with the beta transmembrane subunit, comprising a small extracellular region of 58 amino acids, a transmembrane region of 28 amino acids, and a cytoplasmic tail of 72 amino acids.

   The VNTR regions of the alpha subunit undergo extensive glycosylation, conferring the characteristic mucoid properties of mucin-1. Mucin-1 is expressed in sebaceous glands, ceruminous glands, epithelial cells lining the gastrointestinal and respiratory tracts, mammary glands, vaginal mucosa, and urinary bladder. Within the kidney, mucin-1 is predominantly expressed in the thick ascending limb of the loop of Henle and distal convoluted tubules. Expression has also been reported in leukocytes, including T cells, B cells, monocytes, and dendritic cells.

3. ADTKD-MUC1: Genetics

   Patients with ADTKD-MUC1 carry a pathogenic mutation in one allele of the MUC1 gene, while the second allele remains unaffected. Reported causative mutations occur within the VNTR region and introduce a single-nucleotide frameshift leading to a premature termination codon. In more than 90% of reported cases, the mutation involves a cytosine insertion within a heptanucleotide cytosine tract of the VNTR.

   At the protein level, VNTR units preceding the mutation retain the normal amino acid sequence, whereas downstream repeats acquire an altered reading frame. The N-terminal region and VNTR units preceding the mutation are translated normally. Following the frameshift, the resulting repeat sequences exhibit an approximately 80% reduction in serine and threonine residues, alongside the introduction of one cysteine and six basic amino acid residues per mutated VNTR unit. The resulting frameshift protein (fsMUC1) has a high predicted isoelectric point and is considered potentially toxic to renal tubular epithelial cells.

   Immunohistochemical investigations have demonstrated intracellular accumulation of MUC1-fs in multiple cell types normally expressing mucin-1. Despite the broader tissue distribution of MUC1-fs, clinically significant disease predominantly affects the kidneys. One proposed explanation involves the relatively low turnover of renal tubular epithelial cells, potentially allowing progressive intracellular accumulation of MUC1-fs and subsequent cellular dysfunction and apoptosis. In contrast, epithelial cells in tissues with higher turnover rates, including the skin and gastrointestinal tract, may undergo physiological replacement before comparable accumulation occurs.

   The investigators hypothesize that intracellular accumulation of MUC1-fs contributes directly to the pathogenesis of ADTKD-MUC1. Several observations support the proposed mechanism. Reported pathogenic frameshift mutations generate a common abnormal protein sequence, whereas disease-causing mutations resulting exclusively in truncation or deletion of MUC1 have not been identified. Additionally, MUC1 knockout mice do not exhibit apparent renal abnormalities, suggesting that loss of normal mucin-1 function alone is insufficient to reproduce the disease phenotype. Laboratory investigations have demonstrated cytoplasmic accumulation of MUC1-fs in affected renal tubular cells, while preliminary experimental findings suggest an association between MUC1-fs accumulation and accelerated apoptosis.

   Based on the proposed pathogenic role of MUC1-fs, the investigators hypothesize that reducing MUC1 transcription could decrease production of both wild-type mucin-1 (MUC1) and frameshift mucin-1 (MUC1-fs). Reduced intracellular production and accumulation of MUC1-fs may potentially alleviate renal tubular cellular stress and contribute to preservation of kidney function.

4. Preliminary Data and Rationale: Cholecalciferol Reduces Mucin-1 Expression In Vitro

   Researchers at the Broad Institute, Harvard Medical School, and Massachusetts Institute of Technology developed a high-content screening (HCS) platform using a cell-based immunofluorescence assay to assess the effects of compounds on mucin-1 expression. The screening platform evaluated approximately 4,500 compounds from the Broad Institute drug repurposing library, encompassing compounds at different stages of preclinical and clinical development, including FDA-approved drugs and supplements.

   The primary screen utilized patient-derived cells expressing mutant MUC1 under the endogenous promoter, thereby preserving physiological transcriptional and post-transcriptional regulatory mechanisms. Cholecalciferol was identified as a compound capable of reducing both MUC1 and MUC1-fs protein levels. Immunofluorescence experiments demonstrated decreased intracellular abundance of both proteins following cholecalciferol treatment.

   The promoter sequences of wild-type and mutant MUC1 alleles remain unaffected by the pathogenic VNTR mutation. Consequently, the investigators hypothesize that cholecalciferol may regulate expression of both alleles through shared transcriptional mechanisms, resulting in reduced cellular abundance of wtMUC1 and MUC1-fs.

   The preliminary findings provide a rationale for investigating the effects of cholecalciferol administration on MUC1 and MUC1-fs protein levels in patients with ADTKD-MUC1. Modulation of mucin-1 expression may also have potential relevance to other conditions associated with abnormal mucin-1 expression, including pulmonary interstitial fibrosis and breast cancer.

5. Purpose of the Study

   ADTKD-MUC1 currently has no approved disease-specific treatment. The primary purpose of the study is to investigate the effects of a single high dose of cholecalciferol (vitamin D3) on wild-type and mutant mucin-1 protein levels in Cypriot patients with ADTKD-MUC1.

   The primary outcome is the change in MUC1 and MUC1-fs protein levels measured in urinary extracellular vesicles (EVs) following cholecalciferol administration. The study aims to assess the potential of cholecalciferol to modulate disease-associated protein expression in vivo and generate preliminary evidence supporting further investigation of therapeutic approaches targeting MUC1-fs accumulation.

6. Hypothesis

Administration of a single oral dose of 200,000 IU cholecalciferol is hypothesized to decrease both MUC1 and MUC1-fs protein levels in urinary extracellular vesicles collected from patients with ADTKD-MUC1, with measurable reductions anticipated three days following administration.

The study hypothesis is based on the proposed pathogenic contribution of MUC1-fs accumulation to ADTKD-MUC1 and preliminary in vitro findings demonstrating reduced MUC1 and MUC1-fs abundance following cholecalciferol exposure.

Cholecalciferol (vitamin D3) is commonly administered for the prevention and treatment of vitamin D deficiency. The investigators aim to determine whether administration of a single high dose can reproduce the observed in vitro effects on mucin-1 protein expression in patients with ADTKD-MUC1.

In parallel, collaborating investigators at United States, led by Professor Anthony J. Bleyer, will investigate the effects of ergocalciferol (vitamin D2), following preliminary in vitro experiments demonstrating effects comparable to cholecalciferol.