scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain.

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

Cellular plasticity: A mechanism for homeostasis in the kidney.

Cellular plasticity is a topical subject with interest spanning a wide range of fields from developmental biology to regenerative medicine. Even the nomenclature is a subject of debate, and the underlying mechanisms are still under investigation. On top of injury repair, cell plasticity is a constant physiological process in adult organisms and tissues, in response to homeostatic challenges. In this review we discuss two examples of plasticity for the maintenance of homeostasis in the renal system-namely the renin-producing juxtaglomerular cells (JG cells) and cortical collecting duct (CCD) cells. JG cells show plasticity through recruitment mechanisms, answering the demand for an increase in renin production. In the CCD, cells appear to have the ability to transdifferentiate between principal and intercalated cells to help maintain the highly regulated solute transport levels of that segment. These two cases highlight the complexity of plasticity processes and the role they can play in the kidney.

Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin.

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin-angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d-/- mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d-/- non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system.

Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses.

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P < 0.001; 397 up, 531 down). These clustered in pathways involved in signalling, sterol/lipid metabolism, cell proliferation/hypertrophy and apoptosis. Signalling genes included some implicated in adrenal adenomas but also upregulated genes associated with cyclic AMP and downregulated genes associated with aldosterone synthesis. Sterol metabolism genes were those promoting cholesterol supply (Scarb1, Sqle, Apoa1) and disposal (Cyp27a1, Cyp7b1). Oil red O staining showed lipid depletion consistent with reduced expression of genes involved in lipid synthesis. Genes involved in steroidogenesis (Star, Cyp11a1, Cyp11b1) were modestly affected (P < 0.05; <1.3-fold). Increased Ki67, Ccna2, Ccnb2 and Tk1 expression complemented immunohistochemical evidence of a 3-fold change in cell proliferation. Growth arrest genes, Cdkn1a and Cdkn1c, which are known to be active in hypertrophied cells, were increased >4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu,) were downregulated and apoptotic cells (Tunel staining) were fewer (P < 0.001) and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways.

Zebrafish mesonephric renin cells are functionally conserved and comprise two distinct morphological populations.

Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin (ren), smooth muscle actin (acta2), and platelet-derived growth factor receptor-beta (pdgfrb) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren-expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren-expressing cells.

Renal disease pathophysiology and treatment: contributions from the rat.

The rat has classically been the species of choice for pharmacological studies and disease modeling, providing a source of high-quality physiological data on cardiovascular and renal pathophysiology over many decades. Recent developments in genome engineering now allow us to capitalize on the wealth of knowledge acquired over the last century. Here, we review rat models of hypertension, diabetic nephropathy, and acute and chronic kidney disease. These models have made important contributions to our understanding of renal diseases and have revealed key genes, such as Ace and P2rx7, involved in renal pathogenic processes. By targeting these genes of interest, researchers are gaining a better understanding of the etiology of renal pathologies, with the promised potential of slowing disease progression or even reversing the damage caused. Some, but not all, of these target genes have proved to be of clinical relevance. However, it is now possible to generate more sophisticated and appropriate disease models in the rat, which can recapitulate key aspects of human renal pathology. These advances will ultimately be used to identify new treatments and therapeutic targets of much greater clinical relevance.

Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium.

Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions.

Mineralocorticoid Excess or Glucocorticoid Insufficiency: Renal and Metabolic Phenotypes in a Rat Hsd11b2 Knockout Model.

Obesity and hypertension are 2 major health issues of the 21st century. The syndrome of apparent mineralocorticoid excess is caused by deficiency of 11ß-hydroxysteroid dehydrogenase type 2 (Hsd11b2), which normally inactivates glucocorticoids, rendering the mineralocorticoid receptor aldosterone-specific. The metabolic consequences of Hsd11b2 knockout in the rat are investigated in parallel with electrolyte homeostasis. Hsd11b2 was knocked out, by pronuclear microinjection of targeted zinc-finger nuclease mRNAs, and 1 line was characterized for its response to renal and metabolic challenges. Plasma 11-dehydrocorticosterone was below detection thresholds, and Hsd11b2 protein was undetected by Western blot, indicating complete ablation. Homozygotes were 13% smaller than wild-type littermates, and were polydipsic and polyuric. Their kidneys, adrenals, and hearts were significantly enlarged, but mesenteric fat pads and liver were significantly smaller. On a 0.3% Na diet, mean arterial blood pressure was ≈65 mm Hg higher than controls but only 25 mm Hg higher on a 0.03% Na(+) diet. Urinary Na/K ratio of homozygotes was similar to controls on 0.3% Na(+) diet but urinary albumin and calcium were elevated. Corticosterone and aldosterone levels showed normal circadian variation on both a 0.3% and 0.03% Na(+) diet, but plasma renin was suppressed in homozygotes on both diets. Plasma glucose responses to an oral glucose challenge were reduced despite low circulating insulin, indicating much greater sensitivity to insulin in homozygotes. The rat model reveals mechanisms linking electrolyte homeostasis and metabolic control through the restriction of Hsd11b1 substrate availability.

Hypertrophy in the Distal Convoluted Tubule of an 11ß-Hydroxysteroid Dehydrogenase Type 2 Knockout Model.

Na(+) transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na(+) and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na(+) homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2(-/-) mice, which lack the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na(+) retention in a hypertensive condition. The DCTs of Hsd11b2(-/-) mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11ßHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.

Activation of thiazide-sensitive co-transport by angiotensin II in the cyp1a1-Ren2 hypertensive rat.

Transgenic rats with inducible expression of the mouse Ren2 gene were used to elucidate mechanisms leading to the development of hypertension and renal injury. Ren2 transgene activation was induced by administration of a naturally occurring aryl hydrocarbon, indole-3-carbinol (100 mg/kg/day by gastric gavage). Blood pressure and renal parameters were recorded in both conscious and anesthetized (butabarbital sodium; 120 mg/kg IP) rats at selected time-points during the development of hypertension. Hypertension was evident by the second day of treatment, being preceded by reduced renal sodium excretion due to activation of the thiazide-sensitive sodium-chloride co-transporter. Renal injury was evident after the first day of transgene induction, being initially limited to the pre-glomerular vasculature. Mircoalbuminuria and tubuloinsterstitial injury developed once hypertension was established. Chronic treatment with either hydrochlorothiazide or an AT1 receptor antagonist normalized sodium reabsorption, significantly blunted hypertension and prevented renal injury. Urinary aldosterone excretion was increased ≈ 20 fold, but chronic mineralocorticoid receptor antagonism with spironolactone neither restored natriuretic capacity nor prevented hypertension. Spironolactone nevertheless ameliorated vascular damage and prevented albuminuria. This study finds activation of sodium-chloride co-transport to be a key mechanism in angiotensin II-dependent hypertension. Furthermore, renal vascular injury in this setting reflects both barotrauma and pressure-independent pathways associated with direct detrimental effects of angiotensin II and aldosterone.

Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney.

We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice, and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as were hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in 1) genes involved in vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium (Sgk, Calb1, Trpv5) metabolism associated with calciuria and phosphaturia; 2) genes that would be predicted to desensitize the kidney to glucocorticoid action (Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of enzyme systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggests that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.

Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents.

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11beta-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (aldosterone=0.14%, 11-dehydro-B=0.006%, cortisol=0.016% and DOC=0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y=1.092X-0.054, R(2)=0.988; B ELISA: Y=1.047X-0.226, R(2)=0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.

Angiotensin-converting enzyme is a modifier of hypertensive end organ damage.

Severe forms of hypertension are characterized by high blood pressure combined with end organ damage. Through the development and refinement of a transgenic rat model of malignant hypertension incorporating the mouse renin gene, we previously identified a quantitative trait locus on chromosome 10, which affects malignant hypertension severity and morbidity. We next generated an inducible malignant hypertensive model where the timing, severity, and duration of hypertension was placed under the control of the researcher, allowing development of and recovery from end organ damage to be investigated. We have now generated novel consomic Lewis and Fischer rat strains with inducible hypertension and additional strains that are reciprocally congenic for the refined chromosome 10 quantitative trait locus. We have captured a modifier of end organ damage within the congenic region and, using a range of bioinformatic, biochemical and molecular biological techniques, have identified angiotensin-converting enzyme as the modifier of hypertension-induced tissue microvascular injury. Reciprocal differences between angiotensin-converting enzyme and the anti-inflammatory tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro in the kidney, a tissue susceptible to end organ damage, suggest a mechanism for the amelioration of hypertension-dependent damage.

Cyp11b1 null mouse, a model of congenital adrenal hyperplasia.

Patients with congenital adrenal hyperplasia arising from mutations of 11beta-hydroxylase, the final enzyme in the glucocorticoid biosynthetic pathway, exhibit glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo-pituitary-adrenal activity, and excess mineralocorticoid activity caused by the accumulation of deoxycorticosterone. A mouse model, in which exons 3-7 of Cyp11b1 (the gene encoding 11beta-hydroxylase) were replaced with cDNA encoding enhanced cyan fluorescent protein, was generated to investigate the underlying disease mechanisms. Enhanced cyan fluorescent protein was expressed appropriately in the zona fasciculata of the adrenal gland, and targeted knock-out was confirmed by urinary steroid profiles and, immunocytochemically, by the absence of 11beta-hydroxylase. The null mice exhibited glucocorticoid deficiency, mineralocorticoid excess, adrenal hyperplasia, mild hypertension, and hypokalemia. They also displayed glucose intolerance. Because rodents do not synthesize adrenal androgens, changes in reproductive function such as genital virilization of females were not anticipated. However, adult homozygote females were infertile, their ovaries showing an absence of corpora lutea and a central proliferation of disorganized steroidogenic tissue. Null females responded normally to superovulation, suggesting that raised systemic progesterone levels also contribute to infertility problems. The model reveals previously unrecognized phenotypic subtleties of congenital adrenal hyperplasia.

Identification of a human ortholog of the mouse Dcpp gene locus, encoding a novel member of the CSP-1/Dcpp salivary protein family.

Salivary fluid, the collective product of numerous major and minor salivary glands, contains a range of secretory proteins that play key defensive, digestive, and gustatory roles in the oral cavity. To understand the distinct protein "signature" contributed by individual salivary glands to salivary secretions, we studied a family of proteins shown by in vitro mRNA translation to be abundantly expressed in mouse sublingual glands. Molecular cloning, Southern blotting, and restriction fragment length polymorphism analyses showed these to represent one known and two novel members of the common salivary protein (CSP-1)/Demilune cell and parotid protein (Dcpp) salivary protein family, the genes for which are closely linked in the T-complex region of mouse chromosome 17. Bioinformatic analysis identified a putative human CSP-1/Dcpp ortholog, HRPE773, expressed predominantly in human salivary tissue, that shows 31% amino acid identity and 45% amino acid similarity to the mouse Dcpp query sequence. The corresponding human gene displays a similar structure to the mouse Dcpp genes and is located on human chromosome 16 in a region known to be syntenic with the T-complex region of mouse chromosome 17. The predicted mouse and human proteins both display classical NH(2)-terminal signal sequences, putative jacalin-related lectin domains, and potential N-linked glycosylation sites, suggesting secretion via sublingual saliva into the oral cavity where they may display antimicrobial activity or provide a defensive coating to enamel. Identification of a human CSP-1/Dcpp ortholog therefore provides a key tool for investigation of salivary protein function in human oral health and disease.

Hypertension, kidney, and transgenics: a fresh perspective.

In this review, we outline the application and contribution of transgenic technology to establishing the genetic basis of blood pressure regulation and its dysfunction. Apart from a small number of examples where high blood pressure is the result of single gene mutation, essential hypertension is the sum of interactions between multiple environmental and genetic factors. Candidate genes can be identified by a variety of means including linkage analysis, quantitative trait locus analysis, association studies, and genome-wide scans. To test the validity of candidate genes, it is valuable to model hypertension in laboratory animals. Animal models generated through selective breeding strategies are often complex, and the underlying mechanism of hypertension is not clear. A complementary strategy has been the use of transgenic technology. Here one gene can be selectively, tissue specifically, or developmentally overexpressed, knocked down, or knocked out. Although resulting phenotypes may still be complicated, the underlying genetic perturbation is a starting point for identifying interactions that lead to hypertension. We recognize that the development and maintenance of hypertension may involve many systems including the vascular, cardiac, and central nervous systems. However, given the central role of the kidney in normal and abnormal blood pressure regulation, we intend to limit our review to models with a broadly renal perspective.

Insights from the rat genome sequence.

The availability of the rat genome sequence, and detailed three-way comparison of the rat, mouse and human genomes, is revealing a great deal about mammalian genome evolution. Together with recent developments in cloning technologies, this heralds an important phase in rat research.

Nuclear transfer in rodents.

Cloning is the asexual reproduction of an individual, such that the offspring have an essentially identical nuclear genome. Nuclear transfer and cloning have been achieved in a number of species, namely sheep, cows, goats, rabbits, cats and mice, but have been largely unsuccessful, so far, in dogs, primates and rats. Clearly, contributory factors which affect the outcome of successful cloning experiments are not universally applicable to all species. One theme common to all cloning experiments, however, is the overall inefficiency of the process, typically 0-4%. A number of factors contribute to nuclear transfer inefficiency, and we will review mouse cloning experiments, which address these problems, highlighting the importance of donor nucleus choice (somatic or ES cell, fetal or adult, quiescent or actively dividing). Finally, we will summarize the emerging principles which appear to govern nuclear reprogramming and production of clones, and will consider the application of nuclear transfer to the rat.