Zhang Li

Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2 , expressed in renal epithelial cells, leading to progressive kidney dysfunction and end-stage kidney disease. Patients with PKD exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell RNA sequencing (scRNA-seq) atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Across models, scRNA-seq data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters. Global deletion of Spp1 in Pkd1 ^RC/RC mice resulted in a modest reduction in cyst severity and improved kidney function. From these studies, we created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.

Disruption of PKD2 function causes polycystic kidney disease (PKD) and leads to the formation of cysts in the kidney in both mice and humans. PKD is progressive, initiating with the formation of tubule dilations that expand into large cysts, causing the destruction of renal parenchyma and loss of kidney function. The mechanisms initiating tubule dilation and expansion are uncertain. Tubule obstruction has been proposed as a factor contributing to cystogenesis. Here, we utilize a proximal tubule fluorescent dextran uptake assay to evaluate changes in tubule flow that occur during cyst initiation or expansion and functional decline. We did not observe overt signs of tubule obstruction during cyst initiation or expansion, even in larger cysts; however, the rate of tubule flow within individual nephrons was greatly diminished in cystic kidneys. Surprisingly, along with reduced tubule flow rate, we observe an increase in newly labeled dextran cells associated with disease in the Pkd2 mutants. These cells are spatially distinct from those absorbing the initial dextran and are detected in both cortical and medullary regions of mutant kidneys. Marker analysis indicated that the dextran ⁺ medullary cells were in the loop of Henle and interstitial macrophages. These cells are not present in wild-type controls. This may be due to tubule or vasculature leakage and utilization of renal reserve capacity in mutants that become exhausted, associated with declining glomerular filtration rate (GFR) as cysts expand.

Key Points: A HaloTag knock-in resource allows direct visualization of endogenous polycystin-2 (PC2), enabling quantitative analysis of its localization, turnover, and transport dynamics. PC2-HaloTag labeling establishes PC1-dependent and Tulp3-dependent control of PC2 ciliary targeting in vivo and in cells. Background: Polycystin-2, encoded by PKD2 , is a cation channel essential for kidney physiology. Dysfunction of Pkd2 causes autosomal dominant polycystic kidney disease. Currently, our understanding of cystogenesis in the kidney is limited by the difficulty of visualizing the localization and molecular functions of endogenous polycystin proteins. Methods: Using clustered regularly interspaced short palindromic repeats/Cas9, we engineered a Pkd2 HaloTag knock-in mouse (referred to as Pkd2 ^c-Halo ) and derived tsSV40-immortalized Pkd2 ^c-Halo renal epithelial cell lines. We optimized HaloTag labeling for in vivo and in vitro applications, demonstrating its use in confocal and live cell microscopy, pulse-chase assays, affinity isolation, and in vivo imaging of Pkd2 protein localization after kidney injury and in disease-relevant Tulp3 ^R400W/R400W and Pkd1 ^null mutant backgrounds. Results: Homozygous Pkd2 ^c-Halo mice were viable, fertile, and phenotypically normal, confirming that the C-terminal HaloTag did not disrupt Pkd2 function. Pkd2-c-Halo was detected by Western blotting and localized to endoplasmic reticulum and the primary cilium. Labeling occurred within 30 minutes and plateaued by 3 hours at doses of ≥2 nmol/mouse and ≥25 nM in vivo and in cultured cells, respectively. Pulse-chase analysis showed complete Pkd2-c-Halo turnover within 48 hours in the cilia of the choroid plexus in vivo and 24 hours in cultured renal epithelial cells. HaloTrap affinity resin purified the endogenous Pkd2-c-Halo from cells and tissues efficiently for protein complex analysis. While kidney injury is known to accelerate cyst formation, unilateral ureteral obstruction did not alter Pkd2-c-Halo expression or distribution. Finally, in a Pkd1 null background or in mice homozygous for Tulp3 ^R400W , an allele identified in a patient with hepatorenal cystic disease, Pkd2-c-Halo failed to localize in cilia in kidney tubule epithelium. Conclusions: The Pkd2-c-Halo mouse and derived renal epithelial cell lines enable detection of endogenous polycystin-2 in vivo and in vitro , allowing analysis of its localization, turnover under homeostatic conditions, after kidney injury, and in disease-relevant genetic backgrounds.

Key Points: Macrophage accumulation in cystic kidney disease is not directly regulated by the cilia-dependent cyst activation pathway. Macrophage accumulation and cytokine expression are not driving cyst initiation but rather parallel cyst expansion and contribute to cyst progression. Background: Kidney cyst formation occurs due to loss of cilia-localized polycystin proteins ( e.g ., Pkd1 or Pkd2 ) or ciliary structure ( e.g ., Ift88 or Kif3a ). However, cyst progression is more rapid in polycystin mutant mice compared with cilia mutant mice, and loss of cilia in the polycystin mutant background ( e.g ., Pkd2 and Kif3a mutation) greatly attenuates cyst development. This led to the proposal that the polycystins function to repress a cyst-promoting pathway that is dependent on an intact cilium, this is referred to as the cilia-dependent cyst activation pathway. Renal macrophages are also involved in regulating cyst progression, but it is unknown whether this occurs through the cilia-dependent cyst activation or separate pathway. Methods: To examine whether macrophage accumulation was regulated through a cilia-dependent pathway, we compared macrophage accumulation and cytokine expression levels in Pkd2 mutant kidneys with or without intact cilia ( Ift88 mutants). To avoid the impact of cyst-induced damage on macrophage accumulation, we conducted comparisons after standardizing the samples for cystic indices between the Pkd2 , Ift88 , and Pkd2;Ift88 double mutants. Results: Disruption of Ift88 in Pkd2 mutants reduced cyst burden and attenuated macrophage accumulation and cytokine expression levels. However, when the mutants were standardized based on cystic indices, no significant differences in macrophage number or cytokine expression were evident between the Pkd2 , Ift88 , and Pkd2;Ift88 double mutants at either early or advanced stage of cyst progression, and pathway analysis revealed that the macrophage populations were similar between groups based on single-cell RNAseq data. Conclusions: These data indicated that macrophage accumulation and cytokine expression did not drive cyst initiation but rather paralleled cyst expansion regardless of the genotype or rate of disease progression through a cilia-independent pathway.

Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206+ resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation, and reveal that the CD206+ resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper.

The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney-resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury. While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA-Seq (scRNA-Seq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNA-Seq and spatial transcriptomics revealed 7 distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature, suggesting distinct functions. Specific protein markers were identified for 2 clusters, allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation was lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs was not fully restored for at least 28 days after injury. The change in KRM localization confirmed a long-hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.

Background: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. Methods: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. Results: Analyses of single-cell RNA-seq data of CD45⁺ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. Conclusions: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80 ^hi -expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow–derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII ^– KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.