Autosomal dominant polycystic kidney (ADPKD) represents a common human genetic disease. With an estimated frequency of 1:500 to 1:2000, approximately 80.000 patients are affected in Germany with half of them progressing to end-stage renal disease due to massive cyst growth and renal failure; occasionally, the volume of both kidneys with exceed 10 liters. ADPKD is caused predominantly by mutations in PKD1 (85%) and PKD2 (15%). In addition to the autosomal dominant version, there are many other autosomal recessive cystic kidney diseases that are categorized based on their extra-renal manifestations in different syndromes (e.g. Nephronophthisis (NPHP), Bardet-Biedl syndrome (BBS), etc.).

Most gene products that are mutated in human cystic kidney disease localize to the cilium or affect the function of this microtubular organelle that is attached to most body cells. Although a dysfunction of the cilium has been postulated as the underlying cause of cystic kidney disease (hence the term “ciliopathy”), precise molecular mechanisms of the more than 100 gene products identified so far are still missing.

An intact apical actin cytoskeleton is essential for ciliogenesis. The apical actin cytoskeleton of the multi-ciliated cells of the Xenopus epidermis consists of two layers, the apical and sub-apical actin layer. We found that NPHP family members are essential for formation and maintenance of the sub-apical actin layer.

During early stages of development, the actin cytoskeleton in multi-ciliated cells is highly dynamic, and undergoes rapid assembly and disassembly. After basal body docking to the apical plasma membrane and subsequent cilia formation, the apical actin cytoskeleton becomes progressively more static.

Acute kidney injury (AKI) remains a frequent complication of severe disease. Despite progress in supportive care, the overall mortality and long-term consequences of this complication have not changed significantly. Although complex genetic models and cell lineage tracking have identified subsets of cells that play a central role during the recovery of renal function, the processes that occur immediately after an injury remain unknown because it is virtually impossible to assess these events in mammalian models. Thus, the identification of strategies to prevent AKI and strategies to accelerate recovery has remained an unmet challenge.

We established an approach to elucidate early adaptive changes after kidney damage at the molecular level. Using a workflow, encompassing laser-induced zebrafish kidney injury, high-resolution video-microscopy, single-cell isolation and transcriptional profiling, genetic manipulation of repair genes, and kidney-specific gene knockout in mouse models, we can now analyze early up-stream signaling events and delineate the down-stream metabolic switches occurring in response to kidney injury. Extracting key regulators that orchestrate adaptive responses, we are translating experimental observations into clinical applications. Our strategy aims to change the grim prognosis of AKI by providing novel molecular insight into repair mechanism, while delivering new targets for therapeutic interventions.

To identify the transcriptional changes that occur in response to an injury, we isolate cells directly adjacent to the laser-induced injury.

Although cell ablation by a laser pulse is quite dissimilar to a mammalian IR injury, the cascade of signaling responses that are triggered in surviving cells to initiate the repair process need to entail similar programs: injured cells release signaling molecules to activate programs that allow cells to switch from homeostatic functions to repair programs, including the ability to migrate to the site of the injury. To capture the transcriptional changes occurring in tubular epithelial cells executing the repair, we established single-cell RNA sequencing (scRNAseq) of pronephric duct cells. Transgenic chd17:gfp zebrafish embryos are digested after unilateral pronephros injuries and individual GFP-positive cells rapidly sorted into 384-well plates. RNA from each cell was then extracted, and amplified with bar-coded primers to determine their transcriptome (in collaboration with D. Grün and Sagar at the Max Planck Institute, Freiburg).

1) Immediate (early) responses to injury. The proliferative response, occurring 24-48 hours after injury, has been extensively studied. However, the immediate mechanisms initiating the repair process remain poorly understood, mainly because this process cannot be easily studied in mammalian AKI. Thus, alternative models are needed to identify molecules that orchestrate early adaptive responses after injury.

2) Overall capacity of tubular regeneration. Despite the ability to regenerate, the limits of the regenerative capacity of the kidney remain poorly defined. Although unproven, it is believed the tubular basement membrane (TBM) as guidance cue for regenerating cells needs to remain intact to support the recovery from injury. A better understanding of the role of the TBM may not only help to define the “point of no return” in renal regeneration, but identify new approaches to support the integrity of the TBM.

3) Metabolic adaptation to ischemia. Cells suddenly deprived of oxygen need to switch from aerobic to anaerobic energy production. Although the biochemical pathways are well understood, how this switch is accomplished in vivo is unknown. Manipulating this switch either if AKI is anticipated or after an IR injury has occurred may facilitate recovery from injury.