A small molecule chaperone rescues keratin-8 mediated trafficking of misfolded podocin to correct genetic Nephrotic Syndrome.

Podocin is a key membrane scaffolding protein of the kidney podocyte essential for intact glomerular filtration. Mutations in NPHS2, the podocin-encoding gene, represent the commonest form of inherited nephrotic syndrome (NS), with early, intractable kidney failure. The most frequent podocin gene mutation in European children is R138Q, causing retention of the misfolded protein in the endoplasmic reticulum. Here, we provide evidence that podocin R138Q (but not wild-type podocin) complexes with the intermediate filament protein keratin 8 (K8) thereby preventing its correct trafficking to the plasma membrane. We have also identified a small molecule (c407), a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator protein defect, that interrupts this complex and rescues mutant protein mistrafficking. This results in both the correct localization of podocin at the plasma membrane and functional rescue in both human patient R138Q mutant podocyte cell lines, and in a mouse inducible knock-in model of the R138Q mutation. Importantly, complete rescue of proteinuria and histological changes was seen when c407 was administered both via osmotic minipumps or delivered orally prior to induction of disease or crucially via osmotic minipump two weeks after disease induction. Thus, our data constitute a therapeutic option for patients with NS bearing a podocin mutation, with implications for other misfolding protein disorders. Further studies are necessary to confirm our findings.

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome.

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03-mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9-mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches.

The induction of macrophage hemeoxygenase-1 is protective during acute kidney injury in aging mice.

Aging is thought to be associated with a higher susceptibility to renal ischemia-reperfusion injury (IRI). To study whether defective induction of hemeoxygenase-1 (HO-1, a protective and anti-inflammatory enzyme) might contribute to this, we found that while 12-month-old mice had similar baseline renal function and HO-1 expression, the induction of HO-1 usually seen in ischemia-reperfusion was reduced. This was also associated with worsened renal function and acute tubular necrosis in the aged compared with young mice. In the older mice, heme arginate (HA) induced HO-1 in the cortex and medulla, significantly improved renal function, and reduced tissue injury. Cellular HO-1 induction in the medulla in response to injury or HA treatment was found to be interstitial rather than epithelial, as evidenced by its colocalization with macrophage markers. In vitro, HA treatment of primary macrophages resulted in marked HO-1 induction without impairment of classical activation pathways. Macrophage depletion, caused by diphtheria toxin treatment of 12-month-old CD11b-DTR transgenic animals, resulted in the loss of interstitial HO-1-positive cells and reversal of the protective phenotype of HA treatment. Thus, failure of HO-1 induction following renal IRI worsens structural and functional injury in older mice and represents a therapeutic target in the elderly. Hence, HO-1-positive renal macrophages mediate HA-induced protection in IRI.

Reminder of an important clinical lesson: breast cancer metastasis to the parotid gland.

A 59-year-old woman presented to an otolaryngology clinic with an 8-week history of a painless lump over her left parotid gland. Her medical history included an invasive ductal carcinoma (33 mm) and a ductal carcinoma in situ (70 mm) of the right breast, for which she had a mastectomy and various adjuvant therapies. The primary tumour presented 8 years prior to the metachronous metastasis. This patient was a non-smoker and had no significant family history. Post-superficial parotidectomy pathology revealed the parotid gland tumour to be oestrogen receptor-positive and HER2 receptor-positive, thus ruling out the initial differential diagnosis of a pleomorphic adenoma. A consequential total parotidectomy with a posterolateral neck dissection was performed with sparing of the facial nerve. The patient recovered well having only encountered a self-resolving salivary fistula. She portrayed no signs of facial nerve palsy and subsequent imaging scans showed no abnormalities.