Biology has taught us that a protein as abundantly made and conserved among species as Tamm–Horsfall protein (THP or uromodulin) cannot just be a waste product serving no particular purpose. However, for many researchers, THP is merely a nuisance during urine proteome profiling or exosome purification and for clinicians an enigmatic entity without clear disease implications. Thanks to recent human genetic and correlative studies and animal modeling, we now have a renewed appreciation of this highly prevalent protein in not only guarding urinary homeostasis, but also serving as a critical mediator in systemic inter-organ signaling. Beyond a mere barrier that lines the tubules, or a surrogate for nephron mass, mounting evidence suggests that THP is a multifunctional protein critical for modulating renal ion channel activity, salt/water balance, renal and systemic inflammatory response, intertubular communication, mineral crystallization and bacterial adhesion. Indeed, mutations in THP cause a group of inherited kidney diseases, and altered THP expression is associated with increased risks of urinary tract infection, kidney stone, hypertension, hyperuricemia and acute and chronic kidney diseases. Despite the recent surge of information surrounding THP’s physiological functions and disease involvement, our knowledge remains incomplete regarding how THP is normally regulated by external and intrinsic factors, how precisely THP deficiency leads to urinary and systemic pathophysiology and in what clinical settings THP can be used as a theranostic biomarker and a target for modulation to improve patient outcomes.

Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress. Calcium-dependent protein kinases are plant-like enzymes of apicomplexan parasites that regulate a variety of biological processes including stage-conversion, post-translational repression and egress from the host cell. In this study, we analyzed Toxoplasma CDPK3, which has recently been shown to regulate rapid egress from the host cell. The specific pathways that TgCDPK3 regulates, however, have not previously been known and so we used a quantitative phosphoproteome approach to determine phosphorylation site usage in wild type and TgCDPK3 mutant parasites before, during and after egress. This revealed >150 novel phosphorylation sites that are differentially phosphorylated between WT and TgCDPK3 mutant parasites. Some of these sites are on proteins predicted to play a role in parasite egress. However, we also identified many phosphorylation sites on proteins not thought to be involved in egress, as well as many proteins of unknown function. We confirm that basal calcium levels are affected by CDPK3 inactivation and observed a link between TgCDPK3 and another calcium-dependent kinase (TgCDPK1). Known targets of TgCDPK1 were hyperphosphorylated in the TgCDPK3 mutants, and overexpression of TgCDPK1 partially rescued the observed egress phenotype of TgCDPK3 mutants.

Uromodulin inhibits systemic oxidative stress via TRPM2.

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium‐dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.