In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world\'s most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal readthrough of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation but also limit growth of pre-formed cysts by partially restoring polycystin expression. Furthermore, glycosides accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanisms and future clinical trials.

The Na+-Cl- cotransporter (NCC; SLC12A3) is a highly regulated integral membrane protein that is known to exist as three splice variants in primates. Its primary role in the kidney is to mediate the cosymport of Na+ and Cl- across the apical membrane of the distal convoluted tubule. Through this role and the involvement of other ion transport systems, NCC allows the systemic circulation to reclaim a fraction of the ultrafiltered Na+, K+, Cl-, and Mg+ loads in exchange for Ca2+ and [Formula: see text]. The physiological relevance of the Na+-Cl- cotransport mechanism in humans is illustrated by several abnormalities that result from NCC inactivation through the administration of thiazides or in the setting of hereditary disorders. The purpose of the present review is to discuss the molecular mechanisms and overall roles of Na+-Cl- cotransport as the main topics of interest. On reading the narrative proposed, one will realize that the knowledge gained in regard to these themes will continue to progress unrelentingly no matter how refined it has now become.

Nephrons are the functional units which comprise the kidney. Each nephron contains a number of physiologically unique populations of specialized epithelial cells that are organized into discrete domains known as segments. The principles of nephron segment development have been the subject of many studies in recent years. Understanding the mechanisms of nephrogenesis has enormous potential to expand our knowledge about the basis of congenital anomalies of the kidney and urinary tract (CAKUT), and to contribute to ongoing regenerative medicine efforts aimed at identifying renal repair mechanisms and generating replacement kidney tissue. The study of the zebrafish embryonic kidney, or pronephros, provides many opportunities to identify the genes and signaling pathways that control nephron segment development. Here, we describe recent advances of nephron segment patterning and differentiation in the zebrafish, with a focus on distal segment formation.

Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.

Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells. Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking and monitoring the course of AKI in clinical routine is confined to measuring urine output and plasma levels of filtration markers. Here we demonstrate feasibility and potential of a novel approach to assess the cellular and molecular dynamics of AKI by establishing a robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted kidney cells via flow cytometry sorting. We analyzed 42,608 single cell transcriptomes of 40 urine samples from 32 patients with AKI and compared our data with reference material from human AKI post-mortem biopsies and published mouse data. We demonstrate that tubular epithelial cells transcriptomes mirror kidney pathology and reflect distinct injury and repair processes, including oxidative stress, inflammation, and tissue rearrangement. We also describe an AKI-specific abundant urinary excretion of adaptive progenitor-like cells. Thus, single cell transcriptomics of kidney cells excreted in urine provides noninvasive, unprecedented insight into cellular processes underlying AKI, thereby opening novel opportunities for target identification, AKI sub-categorization, and monitoring of natural disease course and interventions.

Transcellular Mg2+ reabsorption in the distal convoluted tubule (DCT) of the kidneys plays an important role in maintaining systemic Mg2+ homeostasis. SLC41A1 is a Na+/Mg2+ exchanger that mediates Mg2+ efflux from cells and is hypothesized to facilitate basolateral extrusion of Mg2+ in the DCT. In this study, we generated a SLC41A1 knockout mouse model to examine the role of SLC41A1 in Mg2+ homeostasis. Slc41a1-/- mice exhibited similar serum and urine Mg2+ levels as their wild-type littermates. Dietary restriction of Mg2+ resulted in reduced serum Mg2+ concentration and urinary Mg2+ excretion, which was similar in the wild-type and knockout groups. Expression of genes encoding Mg2+ channels and transporters such as transient receptor potential melastatin 6 (Trpm6), transient receptor potential melastatin 7 (Trpm7), cyclin and CBS domain divalent metal cation transport mediator 2 (Cnnm2), and Slc41a3 were unchanged based on genotype. We investigated the potential redundancy of SLC41A1 and its homolog SLC41A3 by generating a double knockout mouse. Although Slc41a3-/- knockout mice showed significantly reduced serum Mg2+ compared with wild-type and Slc41a1-/- knockout groups, double knockout mice displayed similar serum Mg2+ levels as Slc41a3-/- knockout mice. In conclusion, our data show that SLC41A1 is not involved in the regulation of systemic Mg2+ homeostasis in mice. Our data also demonstrate that SLC41A1 does not compensate for the loss of SLC41A3, suggesting different functions of these SLC41 proteins in vivo.NEW & NOTEWORTHY SLC41A1 has been hypothesized to mediate Mg2+ extrusion in the distal convoluted tubule and thus regulate Mg2+ homeostasis. This study investigated the role of SLC41A1 in Mg2+ homeostasis in vivo using a transgenic mouse model. Our results demonstrate that SLC41A1 is not required to maintain normal Mg2+ balance in mice. We also show that SLC41A3 is more important than SLC41A1 in regulating systemic Mg2+ levels.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

BACKGROUND: The proximal tubule (PT) is the major target of cadmium (Cd2+) nephrotoxicity. Current dogma postulates that Cd2+ complexed to metallothionein (MT) (CdMT) is taken up through receptor-mediated endocytosis (RME) via the PT receptor megalin:cubilin, which is the predominant pathway for reuptake of filtered proteins in the kidney. Nevertheless, there is evidence that the distal parts of the nephron are also sensitive to damage induced by Cd2+. In rodent kidneys, another receptor for protein endocytosis, the 24p3 receptor (24p3R), is exclusively expressed in the apical membranes of distal tubules (DT) and collecting ducts (CD). Cell culture studies have demonstrated that RME and toxicity of CdMT and other (metal ion)-protein complexes in DT and CD cells is mediated by 24p3R. In this study, we evaluated the uptake of labeled CdMT complex through 24p3R after acute kidney injury (AKI) induced by gentamicin (GM) administration that disrupts PT function. Subcutaneous administration of GM at 10 mg/kg/day for seven days did not alter the structural and functional integrity of the kidney\'s filtration barrier. However, because of PT injury, the concentration of the renal biomarker Kim-1 increased. When CdMT complex coupled to FITC was administered intravenously, both uptake of the CdMT complex and 24p3R expression in DT increased and also colocalized after PT injury induced by GM. Although megalin decreased in PT after GM administration, urinary protein excretion was not changed, which suggests that the increased levels of 24p3R in the distal nephron could be acting as a compensatory mechanism for protein uptake. Altogether, these results suggest that PT damage increases the uptake of the CdMT complex through 24p3R in DT (and possibly CD) and compensate for protein losses associated with AKI.

Nephronophthisis (NPHP) is a chronic tubular interstitial disorder that exhibits an autosomal recessive genetic form and causes progressive renal failure in children. Patients with NPHP rarely show urinary abnormalities, edema, or hypertension. Thus, NPHP is often detected only when renal failure becomes advanced. NPHP can be divided into three types based on the age of end-stage renal failure, i.e., infant type (approximately 5 years old), juvenile type (approximately 13-14 years old), and adolescent type (approximately 19 years old). Here, we report a case of NPHP diagnosed by genetic analysis at 26 years of age with atypical histological abnormalities.
A 26-year-old woman showed no growth disorders or urinary abnormalities in annual school physical examinations. However, at a check-up at 26 years old, she exhibited renal dysfunction (eGFR 26 mL/min/1.73 m2). Urine tests indicated low specific gravity of urine, but not proteinuria or microscopic hematuria. Urinary β2-microglobulin was high (805 μg/L), and renal biopsy was performed for definitive diagnosis. Histological findings showed no significant findings in glomeruli. However, moderate fibrosis was observed in the interstitial area, and moderate atrophy was observed in the tubules. There were no significant findings in immunofluorescence analysis, and no electron dense deposits were detected by electron microscopy. Although cyst-like expansion of the tubules was unclear, tubular atrophy was dominantly found in the distal tubule by cytokeratin 7 staining. Genetic analysis of the NPHP1 gene showed complete deletion of this gene, leading to a definitive diagnosis of NPHP.
NPHP is not merely a pediatric disease and is relatively high incidence in patients with adult onset end-stage of renal disease. In this case, typical histological abnormalities, such as cyst-like expansion of the tubular lesion, were not observed, and diagnosis was achieved by genetic analysis of the NPHP1 gene, which is responsible for the onset of NPHP. In patients with renal failure with tubular interstitial disease dominantly in the distal tubules, it is necessary to discriminate NPHP, even in adult cases.

UNASSIGNED: Autosomal dominant polycystic kidney disease (ADPKD) causes kidney failure typically in adulthood, but the disease starts in utero. Copeptin, epidermal growth factor (EGF), and monocyte chemoattractant protein-1 (MCP-1) are associated with severity and hold prognostic value in adults but remain unstudied in the early disease stage. Kidneys from adults with ADPKD exhibit macrophage infiltration, and a prominent role of MCP-1 secretion by tubular epithelial cells is suggested from rodent models.
UNASSIGNED: In a cross-sectional study, plasma copeptin, urinary EGF, and urinary MCP-1 were evaluated in a pediatric ADPKD cohort and compared with age-, sex-, and body mass index (BMI)-matched healthy controls. MCP-1 was studied in mouse collecting duct cells, human proximal tubular cells, and fetal kidney tissue.
UNASSIGNED: Fifty-three genotyped ADPKD patients and 53 controls were included. The mean (SD) age was 10.4 (5.9) versus 10.5 (6.1) years (P = 0.543), and the estimated glomerular filtration rate (eGFR) was 122.7 (39.8) versus 114.5 (23.1) ml/min per 1.73 m2 (P = 0.177) in patients versus controls, respectively. Plasma copeptin and EGF secretion were comparable between groups. The median (interquartile range) urinary MCP-1 (pg/mg creatinine) was significantly higher in ADPKD patients (185.4 [213.8]) compared with controls (154.7 [98.0], P = 0.010). Human proximal tubular cells with a heterozygous PKD1 mutation and mouse collecting duct cells with a PKD1 knockout exhibited increased MCP-1 secretion. Human fetal ADPKD kidneys displayed prominent MCP-1 immunoreactivity and M2 macrophage infiltration.
UNASSIGNED: An increase in tubular MCP-1 secretion is an early event in ADPKD. MCP-1 is an early disease severity marker and a potential treatment target.