Genome editing and kidney health.

Genome editing technologies, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas in particular, have revolutionized the field of genetic engineering, providing promising avenues for treating various genetic diseases. Chronic kidney disease (CKD), a significant health concern affecting millions of individuals worldwide, can arise from either monogenic or polygenic mutations. With recent advancements in genomic sequencing, valuable insights into disease-causing mutations can be obtained, allowing for the development of new treatments for these genetic disorders. CRISPR-based treatments have emerged as potential therapies, especially for monogenic diseases, offering the ability to correct mutations and eliminate disease phenotypes. Innovations in genome editing have led to enhanced efficiency, specificity and ease of use, surpassing earlier editing tools such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs). Two prominent advancements in CRISPR-based gene editing are prime editing and base editing. Prime editing allows precise and efficient genome modifications without inducing double-stranded DNA breaks (DSBs), while base editing enables targeted changes to individual nucleotides in both RNA and DNA, promising disease correction in the absence of DSBs. These technologies have the potential to treat genetic kidney diseases through specific correction of disease-causing mutations, such as somatic mutations in PKD1 and PKD2 for polycystic kidney disease; NPHS1, NPHS2 and TRPC6 for focal segmental glomerulosclerosis; COL4A3, COL4A4 and COL4A5 for Alport syndrome; SLC3A1 and SLC7A9 for cystinuria and even VHL for renal cell carcinoma. Apart from editing the DNA sequence, CRISPR-mediated epigenome editing offers a cost-effective method for targeted treatment providing new avenues for therapeutic development, given that epigenetic modifications are associated with the development of various kidney disorders. However, there are challenges to overcome, including developing efficient delivery methods, improving safety and reducing off-target effects. Efforts to improve CRISPR-Cas technologies involve optimizing delivery vectors, employing viral and non-viral approaches and minimizing immunogenicity. With research in animal models providing promising results in rescuing the expression of wild-type podocin in mouse models of nephrotic syndrome and successful clinical trials in the early stages of various disorders, including cancer immunotherapy, there is hope for successful translation of genome editing to kidney diseases.

Podocyte protease activated receptor 1 stimulation in mice produces focal segmental glomerulosclerosis mirroring human disease signaling events.

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/-) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6.

Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation.

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.