Autosomal dominant tubulointerstitial kidney disease (ADTKD), and ADTKD-MUC1, continues to present a significant diagnostic challenge for nephrologists. The autosomal dominant inheritance pattern is characteristic, but not much else is. Those affected present with progressive CKD later in life and end stage kidney disease (ESKD) can occur anytime between the ages of 17 and 75. Urinalysis is bland and kidney biopsies tend to show interstitial scarring and no evidence of inflammation or glomerular disease, but no distinctive characteristic features. Kidney ultrasound is also often unremarkable, and it is this that prompted a KDIGO consensus guideline to recommend a name change from medullary cystic kidney disease to ADTKD in 2015. Cysts are neither pathognomonic for the disease, nor are they necessarily confined to the medulla when present. ADTKD-MUC1 also tends to lack the characteristic extra-renal features of other forms of ADTKD - hyperuricemia and gout in those with ADTKD-UMOD, hyperuricemia and anemia in ADTKD-REN and diabetes and deranged liver function in ADTKD-HNF1B.
To compound this diagnostic challenge, MUC1 is an extremely challenging gene to sequence. Most families with ADTKD-MUC1 have a single cytosine(c) insertion into a string of seven c base pairs in a variable nucleotide tandem repeat (VNTR) sequence in MUC1. This insertion leads to a frameshift mutation and the formation of a mutant protein, MUC1-fs, which is responsible for ADTKD-MUC1. A VNTR is a short sequence of nucleotides organised into a tandem repeat. In this case the VNTR codes for a serine, proline, and threonine rich sequence of 20 amino acids that make up the extracellular part of the Mucin-1 protein. The VNTR is high in cytosine and guanine (GC) residues, making it more difficult to denature and therefore more difficult to sequence. For these reasons, MUC1 is refractory to many next generation sequencing methods and a novel mass spectrometer-based method has instead been developed to detect the C+ insertion in the VNTR. The single C+ insertion destroys a restriction site cleaved by the endonuclease Mwol. Mwol is used to cleave the wild type, leaving the C+ units intact. This allows enzymatic enrichment of the mutant signal. Mass spectrometry is then used to distinguish signal intensity. This test will not detect any novel mutations in MUC1.
This sequencing method is not currently commercially available, and can only be performed in one centre worldwide, the Broad Institute, Massachusetts, via Wake Forest Health, so reproducibility was an issue. However, recently other immunohistological methods of staining for the MUC1-fs protein in urine and on kidney biopsy have become available. These have been able to detect the MUC1-fs protein not only in those with C+ insertions but also other novel mutations.
MUC1 codes for the protein, Mucin-1. Mucins are a form of high molecular weight proteins produced by epithelial cells, which are capable of forming gels. They line epithelial surfaces and help protect against pathogens. Mucin-1 has an extracellular component and a transmembrane domain that spans the apical surface of epithelial cells in the kidney, mammary gland, lung, digestive tract, prostate and uterus. It is this extra-cellular component which contains the sequence of 20 amino acids coded for by the VNTR in which pathogenic MUC1 mutations largely occur. The formation of MUC1-fs protein is integral to the occurrence of ADTKD-MUC1, and to date, no families have been identified that have ADTKD-MUC1 with a different protein or a truncated version of that protein. This peptide lacks the transmembrane and extracellular components of Mucin-1 and carries a very high positive charge. It deposits intracellularly. Though it deposits in cells throughout the body, it’s clinically harmful effects appear to be limited to the kidney. In the kidney it leads to accelerated apoptosis, tubular damage and progressive CKD.
In this study the authors were able to generate mouse models and organoids that replicated the behaviour of wild type (WT) and MUC1-fs. MUC1-wt was expressed on the apical membrane, while MUC1-fs was expressed intracellularly. Both developed changes similar to those seen on renal biopsies of families with ADTKD-MUC1. The study looked at the effect of the unfolded protein response (UPR) in ADTKD-MUC1.The UPR is a stress response that occurs in response to the presence of misfolded or unfolded proteins in the endoplasmic reticulum. It acts to halt translation, degrade misfolded proteins and if homeostasis cannot be restored, will induce apoptosis. The study showed that in the presence of a fully functional UPR, MUC1-fs protein accumulation alone is not enough to cause cell damage, but that if the ATF6 (activating transcription factor 6) portion of the pathway was lost, this did lead to increased cell apoptosis. They hypothesise that while ATF6 is functional it can clear MUC1-fs, but that as additional insults, such as exposure to inflammation, infections and nephrotoxins, occur throughout life these additional stresses may activate the other apoptotic pathways of the UPR. This may account for the variation in age of onset of CKD and ESRD.
They screened upwards of 3,000 compounds and identified one, BRD4780, that preferentially removed MUC1-fs in a dose dependent manner, without affecting either MUC1-wt or impairing MUC1 transcription. BRD4780 appeared to rescue MUC1-fs affected cells from cell death, while reducing UPR activation. When tested on mouse-models and in vivo it appeared to clear the mutant protein from intracellular compartments while MUC1-wt levels remained unchanged.
The secretory pathway is a pathway by which nuclear encoded mRNAs are translated into proteins and transferred to the lysosome or cell membrane or excreted from the cells. Nuclear-encoded mRNAs undergo translation on cytosolic ribosomes after which an endoplasmic reticulum (ER) signal sequence directs them to the ER. Translation is completed to the ER and then the proteins are moved via transport vesicles to the Golgi apparatus. There they are sorted and delivered to several destinations including secretion or placement in the cell membrane. In this paper they performed a co-localisation study and determined that MUC1-fs was becoming trapped in the early secretory pathway, and not progressing to the membrane. MUC1-fs tended to stay in the Golgi compartment and TMED9 cargo receptor-positive vesicles. TMED9 appears to be responsible for packaging and transport MUC1-fs from the ER to the Golgi compartment and also in retrograde transport back to the ER. Evidence from mouse models, organoids and human cells suggests that MUC1-fs is distributed with TMED9. BRD4780 appears to release MUC1-fs from the early secretory compartment and allow it to reach the lysosome where it can be broken down.BRD4780 appears to bind to TMED9 directly, leading to the release of MUC1-fs out of the early secretory pathway, though the mechanism of action of this has yet to be elucidated.
BRD4780 also showed promise in removing other early secretory pathway proteins, reducing intracellular levels of mutant UMOD and of rhodopsin, which causes retinitis pigmentosa. It had no affect on huntington, which causes Huntington’s disease.
In summary, ADTKD-MUC1 is a rare form of kidney disease and poses significant clinical and genetic diagnostic challenges. The most important element in diagnosis is a thorough family history. Promising new modalities of diagnosis and treatment are beginning to open up for this rare disease. BRD4780 shows promise in the treatment of not only MUC1-fs but in other proteinopathies, if pre-clinical trials can be translated into clinical success.
Commentary by Susan Murray
Nephrology Fellow, Royal College of Surgeons, Ireland