Kidney Nephron Determination.

The nephron is a multifunctional filtration device equipped with an array of sophisticated sensors. For appropriate physiological function in the human and mouse, nephrons must be stereotypically arrayed in large numbers, and this essential structural property that defines the kidney is determined during its fetal development. This review explores the process of nephron determination in the fetal kidney, providing an overview of the foundational literature in the field as well as exploring new developments in this dynamic research area. Mechanisms that ensure that a large number of nephrons can be formed from a small initial number of progenitor cells are central to this process, and the question of how the nephron progenitor cell population balances epithelial differentiation with renewal in the progenitor state is a subject of particular interest. Key growth factor signaling pathways and transcription factor networks are discussed.

Bioenergetics: the evolutionary basis of progressive kidney disease.

Chronic kidney disease (CKD) affects >10% of the world population, with increasing prevalence in middle age. The risk for CKD is dependent on the number of functioning nephrons through the life cycle, and 50% of nephrons are lost through normal aging, revealing their vulnerability to internal and external stressors. Factors responsible for CKD remain poorly understood, with limited availability of biomarkers or effective therapy to slow progression. This review draws on the disciplines of evolutionary medicine and bioenergetics to account for the heterogeneous nephron injury that characterizes progressive CKD following episodes of acute kidney injury with incomplete recovery. The evolution of symbiosis in eukaryotes led to the efficiencies of oxidative phosphorylation and the rise of metazoa. Adaptations to ancestral environments are the products of natural selection that have shaped the mammalian nephron with its vulnerabilities to ischemic, hypoxic, and toxic injury. Reproductive fitness rather than longevity has served as the driver of evolution, constrained by available energy and its allocation to homeostatic responses through the life cycle. Metabolic plasticity has evolved in parallel with robustness necessary to preserve complex developmental programs, and adaptations that optimize survival through reproductive years can become maladaptive with aging, reflecting antagonistic pleiotropy. Consequently, environmental stresses promote trade-offs and mismatches that result in cell fate decisions that ultimately lead to nephron loss. Elucidation of the bioenergetic adaptations by the nephron to ancestral and contemporary environments may lead to the development of new biomarkers of kidney disease and new therapies to reduce the global burden of progressive CKD.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis.

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Aberrant centrosome biogenesis disrupts nephron and collecting duct progenitor growth and fate resulting in fibrocystic kidney disease.

Mutations that disrupt centrosome biogenesis or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet how centrosome dysfunction results in the kidney disease phenotypes remains unknown. Here, we examined the consequences of conditional knockout of the ciliopathy gene Cep120, essential for centrosome duplication, in the nephron and collecting duct progenitor niches of the mouse embryonic kidney. Cep120 loss led to reduced abundance of both cap mesenchyme and ureteric bud populations, due to a combination of delayed mitosis, increased apoptosis and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis and decline in kidney function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in the pathways essential for development, fibrosis and cystogenesis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney morphogenesis and identifies new therapeutic targets for patients with renal centrosomopathies.

Impaired cell fate through gain-of-function mutations in a chromatin reader.

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.

Isthmin-1: A critical regulator of branching morphogenesis and metanephric mesenchyme condensation during early kidney development.

Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8ß1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.

Overgrowth syndromes and pediatric cancers: how many roads lead to IGF2?

Overgrowth syndromes such as Perlman syndrome and associated pediatric cancers, including Wilms tumor, arise through genetic and, in certain instances, also epigenetic changes. In the case of the Beckwith-Wiedemann overgrowth syndrome and in Wilms tumor, increased levels of IGF2 have been shown to be causally related to the disease manifestation. In the previous issue of Genes & Development, Hunter and colleagues (pp. 903-908) investigated the molecular mechanisms by which mutations in the gene encoding the RNA degradation component DIS3L2 lead to Perlman syndrome. By analyzing nephron progenitor cells derived from their newly created Dis3l2 mutant mouse lines, the investigators showed that DIS3L2 loss of function leads to up-regulation of IGF2 independently of the let7 microRNA pathway. In a second study in this issue of Genes & Development, Chen and colleagues (pp. 996-1007) show that microRNA processing gene mutations in Wilms tumor lead to an increase in the levels of transcription factor pleomorphic adenoma gene 1 (PLAG1) that in turn activates IGF2 expression. Thus, augmented IGF2 expression seems to be a common downstream factor in both tissue overgrowth and Wilms tumor through several alternative mechanisms.

Loss of Dis3l2 partially phenocopies Perlman syndrome in mice and results in up-regulation of Igf2 in nephron progenitor cells.

Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.

Bmp7 drives proximal tubule expansion and determines nephron number in the developing kidney.

The mammalian kidney is composed of thousands of nephrons that are formed through reiterative induction of a mesenchymal-to-epithelial transformation by a population of nephron progenitor cells. The number of nephrons in human kidneys ranges from several hundred thousand to nearly a million, and low nephron number has been implicated as a risk factor for kidney disease as an adult. Bmp7 is among a small number of growth factors required to support the proliferation and self-renewal of nephron progenitor cells, in a process that will largely determine the final nephron number. Once induced, each nephron begins as a simple tubule that undergoes extensive proliferation and segmental differentiation. Bmp7 is expressed both by nephron progenitor cells and the ureteric bud derivative branches that induce new nephrons. Here, we show that, in mice, Bmp7 expressed by progenitor cells has a major role in determining nephron number; nephron number is reduced to one tenth its normal value in its absence. Postnatally, Bmp7 also drives proliferation of the proximal tubule cells, and these ultimately constitute the largest segment of the nephron. Bmp7 appears to act through Smad 1,5,9(8), p38 and JNK MAP kinase. In the absence of Bmp7, nephrons undergo a hypertrophic process that involves p38. Following a global inactivation of Bmp7, we also see evidence for Bmp7-driven growth of the nephron postnatally. Thus, we identify a role for Bmp7 in supporting the progenitor population and driving expansion of nephrons to produce a mature kidney.

FGF8 induces chemokinesis and regulates condensation of mouse nephron progenitor cells.

Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.

Revisiting the role of Notch in nephron segmentation confirms a role for proximal fate selection during mouse and human nephrogenesis.

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human induced pluripotent stem cells (iPSCs) to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the demonstration that nicastrin (NCSTN)-deficient human iPSC-derived organoids differentiate into TFA2B+ distal tubule and CDH1+ connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism.

Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.

Empagliflozin in Heart Failure: Regional Nephron Sodium Handling Effects.

SIGNIFICANCE STATEMENT: The effect of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on regional tubular sodium handling is poorly understood in humans. In this study, empagliflozin substantially decreased lithium reabsorption in the proximal tubule (PT) (a marker of proximal tubular sodium reabsorption), a magnitude out of proportion to that expected with only inhibition of sodium-glucose cotransporter-2. This finding was not driven by an "osmotic diuretic" effect; however, several parameters changed in a manner consistent with inhibition of the sodium-hydrogen exchanger 3. The large changes in proximal tubular handling were acutely buffered by increased reabsorption in both the loop of Henle and the distal nephron, resulting in the observed modest acute natriuresis with these agents. After 14 days of empagliflozin, natriuresis waned due to increased reabsorption in the PT and/or loop of Henle. These findings confirm in humans that SGLT2i have complex and important effects on renal tubular solute handling. BACKGROUND: The effect of SGLT2i on regional tubular sodium handling is poorly understood in humans but may be important for the cardiorenal benefits. METHODS: This study used a previously reported randomized, placebo-controlled crossover study of empagliflozin 10 mg daily in patients with diabetes and heart failure. Sodium handling in the PT, loop of Henle (loop), and distal nephron was assessed at baseline and day 14 using fractional excretion of lithium (FELi), capturing PT/loop sodium reabsorption. Assessments were made with and without antagonism of sodium reabsorption through the loop using bumetanide. RESULTS: Empagliflozin resulted in a large decrease in sodium reabsorption in the PT (increase in FELi=7.5%±10.6%, P = 0.001), with several observations suggesting inhibition of PT sodium hydrogen exchanger 3. In the absence of renal compensation, this would be expected to result in approximately 40 g of sodium excretion/24 hours with normal kidney function. However, rapid tubular compensation occurred with increased sodium reabsorption both in the loop ( P < 0.001) and distal nephron ( P < 0.001). Inhibition of sodium-glucose cotransporter-2 did not attenuate over 14 days of empagliflozin ( P = 0.14). However, there were significant reductions in FELi ( P = 0.009), fractional excretion of sodium ( P = 0.004), and absolute fractional distal sodium reabsorption ( P = 0.036), indicating that chronic adaptation to SGLT2i results primarily from increased reabsorption in the loop and/or PT. CONCLUSIONS: Empagliflozin caused substantial redistribution of intrarenal sodium delivery and reabsorption, providing mechanistic substrate to explain some of the benefits of this class. Importantly, the large increase in sodium exit from the PT was balanced by distal compensation, consistent with SGLT2i excellent safety profile. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov ( NCT03027960 ).

Kidney development to kidney organoids and back again.

Kidney organoid technology has led to a renaissance in kidney developmental biology. The complex underpinnings of mammalian kidney development have provided a framework for the generation of kidney cells and tissues from human pluripotent stem cells. Termed kidney organoids, these 3-dimensional structures contain kidney-specific cell types distributed similarly to in vivo architecture. The adult human kidney forms from the reciprocal induction of two disparate tissues, the metanephric mesenchyme (MM) and ureteric bud (UB), to form nephrons and collecting ducts, respectively. Although nephrons and collecting ducts are derived from the intermediate mesoderm (IM), their development deviates in time and space to impart distinctive inductive signaling for which separate differentiation protocols are required. Here we summarize the directed differentiation protocols which generate nephron kidney organoids and collecting duct kidney organoids, making note of similarities as much as differences. We discuss limitations of these present approaches and discuss future directions to improve kidney organoid technology, including a greater understanding of anterior IM and its derivatives to enable an improved differentiation protocol to collecting duct organoids for which historic and future developmental biology studies will be instrumental.

Rap1 regulates lumen continuity via Afadin in renal epithelia.

The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Lectin-mediated, time-efficient, and high-yield sorting of different morphologically intact nephron segments.

The kidney is a highly complex organ equipped with a multitude of miniscule filter-tubule units called nephrons. Each nephron can be subdivided into multiple segments, each with its own morphology and physiological function. To date, conventional manual approaches to isolate specific nephron segments are very laborious, time-consuming, often limited to only a specific segment, and typically have low yield. Here, we describe a novel, unconventional method that is superior in many aspects to previous protocols by combining low-cost fluorophore-conjugated lectins or agglutinins (Flaggs) with flow sorting. This allows the simultaneous separation of different nephron segments with preserved 3D morphology from mouse or human samples in under 3 h. Using a 200-µm nozzle and 5 psi, glomeruli, proximal, or distal convoluted tubules are sorted with Cy3-labeled Sambucus Nigra agglutinin (SNA-Cy3), Fluorescein-labeled Lotus Tetragonolobus lectin (LTL-FITC), or Pacific Blue-labeled soybean agglutinin (SBA-PB), respectively. Connecting tubules and collecting ducts are sorted by double-positive SBA-PB and SNA-Cy3 signals, while thick ascending limb segments are characterized by the absence of any Flaggs labeling. From two mouse kidneys, this yields 37-521 ng protein/s or 0.71-16.71 ng RNA/s, depending on the specific nephron segment. The purity of sorted segments, as assessed by mRNA expression level profiling of 15 genes, is very high with a 96.1-fold median enrichment across all genes and sorted segments. In summary, our method represents a simple, straightforward, cost-effective, and widely applicable tool yielding high amounts of pure and morphologically largely intact renal tubule materials with the potential to propel nephron segment-specific research.

Pendrin: linking acid base to blood pressure.

Pendrin (SLC26A4) is an anion exchanger from the SLC26 transporter family which is mutated in human patients affected by Pendred syndrome, an autosomal recessive disease characterized by sensoneurinal deafness and hypothyroidism. Pendrin is also expressed in the kidney where it mediates the exchange of internal HCO3- for external Cl- at the apical surface of renal type B and non-A non-B-intercalated cells. Studies using pendrin knockout mice have first revealed that pendrin is essential for renal base excretion. However, subsequent studies have demonstrated that pendrin also controls chloride absorption by the distal nephron and that this mechanism is critical for renal NaCl balance. Furthermore, pendrin has been shown to control vascular volume and ultimately blood pressure. This review summarizes the current knowledge about how pendrin is linking renal acid-base regulation to blood pressure control.

Angiotensin II-stimulated proximal nephron superoxide production and fructose-induced salt-sensitive hypertension.

Angiotensin II (ANG II) increases proximal tubule superoxide (O2-) production more in rats fed a 20% fructose normal-salt diet compared with rats fed a 20% glucose normal-salt diet. A 20% fructose high-salt diet (FHS) increases systolic blood pressure (SBP), whereas a 20% glucose high-salt diet (GHS) does not. However, it is unclear whether FHS enhances ANG II-induced oxidative stress in proximal tubules and whether this contributes to increases in blood pressure in this model. We hypothesized that FHS augments the ability of ANG II to stimulate O2- production by proximal tubules, and this contributes to fructose-induced salt-sensitive hypertension. We measured SBP in male Sprague-Dawley rats fed FHS and GHS and determined the effects of 3 mM tempol and 50 mg/kg losartan for 7 days. We then measured basal and ANG II-stimulated (3.7 × 10-8 M) O2- production by proximal tubule suspensions and the role of protein kinase C. FHS increased SBP by 27 ± 5 mmHg (n = 6, P < 0.006) but GHS did not. Rats fed FHS + tempol and GHS + tempol showed no significant increases in SBP. ANG II increased O2- production by 11 ± 1 relative light units/µg protein/s in proximal tubules from FHS-fed rats (n = 6, P < 0.05) but not in tubules from rats fed GHS. ANG II did not significantly stimulate O2- production by proximal tubules from rats fed FHS + tempol or FHS + losartan. The protein kinase C inhibitor Gö6976 blunted ANG II-stimulated O2- production. In conclusion, FHS enhances the sensitivity of proximal tubule O2- production to ANG II, and this contributes to fructose-induced salt-sensitive hypertension.NEW & NOTEWORTHY A diet containing amounts of fructose consumed by 17 million Americans causes salt-sensitive hypertension. Oxidative stress is an initiating cause of this model of fructose-induced salt-sensitive hypertension increasing blood pressure. This salt-sensitive hypertension is prevented by losartan and thus is angiotensin II (ANG II) dependent. Fructose-induced salt-sensitive hypertension depends on ANG II stimulating oxidative stress in the proximal tubule. A fructose/high-salt diet augments the ability of ANG II to stimulate proximal tubule O2- via protein kinase C.