Search

The kidney has an extraordinary ability to maintain glomerular filtration despite natural fluctuations in blood pressure and nephron loss. This is partly due to local coordination between single-nephron filtration and vascular perfusion. An improved understanding of the three-dimensional (3-D) functional coordination between nephrons and the vasculature may provide a new perspective of the heterogeneity of kidney function and could inform targeted therapies and timed interventions to slow or prevent the progression of kidney disease. Here, we developed magnetic resonance imaging (MRI) tools to visualize single-nephron function in 3-D throughout the isolated perfused rat kidney. We used an intravenous slow perfusion of a glomerulus-targeted imaging tracer [cationized ferritin (CF)] to map macromolecular dynamics and to identify glomeruli in 3-D, followed by a bolus of a freely filtered tracer (gadolinium diethylenetriamine penta-acetic acid) to map filtration kinetics. There was a wide intrakidney distribution of CF binding rates and estimated single-nephron glomerular filtration rate (eSNGFR) between nephrons. eSNGFR and CF uptake rates did not vary significantly by distance from the kidney surface. eSNGFR varied from ∼10 to ∼100 nL/min throughout the kidney. Whole single-kidney GFR was similar across all kidneys, despite differences in the distributions eSNGFR of and glomerular number, indicating a robust adaptive regulation of individual nephrons to maintain constant single-kidney GFR in the presence of a natural variation in nephron number. This work provides a framework for future studies of single-nephron function in the whole isolated perfused kidney and experiments of single-nephron function in vivo using MRI.

Based on analysis of 819 human biopsies, essential derangement in diabetic nephropathy consists of accumulation of worn-out glomerular basement membrane in the mesangium that may advance to global sclerosis. The most frequent pathway to nephron dropout starts with the penetration of glomerular capillaries into Bowman’s capsule (BC), delivering an exudate into BC that spreads around the entire glomerular circumference and via the glomerulotubular junction onto the tubule, resulting in glomerular sclerosis and chronic tubulointerstitial damage.

Kidney pathologies are often highly heterogeneous. To comprehensively understand kidney structure and pathology, it is critical to develop tools to map tissue microstructure in the context of the whole, intact organ. Magnetic resonance imaging (MRI) can provide a unique, three-dimensional view of the kidney and allows for measurements of multiple pathological features. Here, we developed a platform to systematically render and map gross and microstructural features of the human kidney based on three-dimensional MRI. These features include pyramid number and morphology as well as the associated medulla and cortex. In a subset of these kidneys, we also mapped individual glomeruli and glomerular volumes using cationic ferritin-enhanced MRI to report intrarenal heterogeneity in glomerular density and size. Finally, we rendered and measured regions of nephron loss due to pathology and individual glomerular volumes in each pyramidal unit. This work provides new tools to comprehensively evaluate the kidney across scales, with potential applications in anatomic and physiological research, transplant allograft evaluation, biomarker development, biopsy guidance, and therapeutic monitoring. These image rendering and analysis tools could eventually impact the field of transplantation medicine to improve longevity matching of donor allografts and recipients and reduce discard rates through the direct assessment of donor kidneys. NEW & NOTEWORTHY We report the application of cutting-edge image analysis approaches to characterize the pyramidal geometry, glomerular microstructure, and heterogeneity of the whole human kidney imaged using MRI. This work establishes a framework to improve the detection of microstructural pathology to potentially facilitate disease monitoring or transplant evaluation in the individual kidney.

Large-conductance K(+) (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K(+) secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K(+) diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K(+) diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K(+). We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K(+) diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K(+) (5% K(+), as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K(+) excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K(+) diet have higher blood K(+) concentration and reduced IC BK channel activity are consistent with impaired urinary K(+) secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K(+) diet. NEW & NOTEWORTHY When mice are placed on a high-K(+) diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K(+) channel, blunted large-conductance K(+) channel currents in intercalated cells, and increased blood K(+) concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K(+) diet.

The renal nephron consists of a series of distinct cell types that function in concert to maintain fluid and electrolyte balance and blood pressure. The renin-angiotensin system (RAS) is central to Na(+) and volume balance. We aimed to determine how loss of angiotensin II signaling in the proximal tubule (PT), which reabsorbs the bulk of filtered Na(+) and volume, impacts solute transport throughout the nephron. We hypothesized that PT renin-angiotensin system disruption would not only depress PT Na(+) transporters but also impact downstream Na(+) transporters. Using a mouse model in which the angiotensin type 1a receptor (AT(1a)R) is deleted specifically within the PT (AT(1a)R PTKO), we profiled the abundance of Na(+) transporters, channels, and claudins along the nephron. Absence of PT AT(1a)R signaling was associated with lower abundance of PT transporters (Na(+)/H(+) exchanger isoform 3, electrogenic Na(+)-bicarbonate cotransporter 1, and claudin 2) as well as lower abundance of downstream transporters (total and phosphorylated Na(+)-K(+)-2Cl(−) cotransporter, medullary Na(+)-K(+)-ATPase, phosphorylated NaCl cotransporter, and claudin 7) versus controls. However, transport activities of Na(+)-K(+)-2Cl(−) cotransporter and NaCl cotransporter (assessed with diuretics) were similar between groups in order to maintain electrolyte balance. Together, these results demonstrate the primary impact of angiotensin II regulation on Na(+) reabsorption in the PT at baseline and the associated influence on downstream Na(+) transporters, highlighting the ability of the nephron to integrate Na(+) transport along the nephron to maintain homeostasis. NEW & NOTEWORTHY Our study defines a novel role for proximal tubule angiotensin receptors in regulating the abundance of Na(+) transporters throughout the nephron, thereby contributing to the integrated control of fluid balance in vivo.

Kidney transport and other renal functions are regulated by multiple G protein-coupled receptors (GPCRs) expressed along the renal tubule. The rapid, recent appearance of comprehensive unbiased gene expression data in the various renal tubule segments, chiefly RNA sequencing and protein mass spectrometry data, has provided a means of identifying patterns of GPCR expression along the renal tubule. To allow for comprehensive mapping, we first curated a comprehensive list of GPCRs in the genomes of mice, rats, and humans ( https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/ ) using multiple online data sources. We used this list to mine segment-specific and cell type-specific expression data from RNA-sequencing studies in microdissected mouse tubule segments to identify GPCRs that are selectively expressed in discrete tubule segments. Comparisons of these mapped mouse GPCRs with other omics datasets as well as functional data from isolated perfused tubule and micropuncture studies confirmed patterns of expression for well-known receptors and identified poorly studied GPCRs that are likely to play roles in the regulation of renal tubule function. Thus, we provide data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors to provide guidance for future studies.

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K(+) excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca(2+)-activated large-conductance K(+) channel subunit-α (BKα), ENaCβ, ENaCγ, total NCC, and phospho-NCC (at Thr(53)) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l(fl/fl)) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4l(flox/flox) mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K(+) excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4l(flox/flox) mice. In contrast, plasma Na(+) concentrations were similar in Nedd4l(flox/flox) and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K(+) excretion and plasma K(+) ranges during long-term HS diet. NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na(+) channel activity and that Nedd4-2 plays an essential role in maintaining K(+) homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic ( Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic ( Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type ( Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

Angiotensin II exacerbates oxidative stress in part by increasing superoxide ([Formula: see text]) production by many renal tissues. However, whether it does so in proximal tubules and the source of [Formula: see text] in this segment are unknown. Dietary fructose enhances the stimulatory effect of angiotensin II on proximal tubule Na+reabsorption, but whether this is true for oxidative stress is unknown. We hypothesized that angiotensin II causes proximal nephron oxidative stress in part by stimulating NADPH oxidase (NOX)4-dependent [Formula: see text] production and decreasing the amount of the antioxidant glutathione, and this is exacerbated by dietary fructose. We measured basal and angiotensin II-stimulated [Formula: see text] production with and without inhibitors, NOX1 and NOX4 expression, and total and reduced glutathione (GSH) in proximal tubules from rats drinking either tap water (control) or 20% fructose. Angiotensin II (10 nM) increased [Formula: see text] production by 113 ± 42 relative light units·mg protein−1·s−1in controls and 401 ± 74 relative light units·mg protein−1·s−1with 20% fructose ( n = 11 for each group, P < 0.05 vs. control). Apocynin and the Nox1/4 inhibitor GKT136901 prevented angiotensin II-induced increases in both groups. NOX4 expression was not different between groups. NOX1 expression was undetectable. Angiotensin II decreased GSH by 1.8 ± 0.8 nmol/mg protein in controls and by 4.2 ± 0.9 nmol/mg protein with 20% fructose ( n = 18 for each group, P < 0.047 vs. control). We conclude that 1) angiotensin II causes oxidative stress in proximal tubules by increasing [Formula: see text] production by NOX4 and decreasing GSH and 2) dietary fructose enhances the ability of angiotensin II to stimulate [Formula: see text] and diminish GSH, thereby exacerbating oxidative stress in this segment.

Randall’s plaque (RP; subepithelial calcification) appears to be an important precursor of kidney stone disease. However, RP cannot be noninvasively detected. The present study investigated candidate biomarkers associated with extracellular vesicles (EVs) in the urine of calcium stone formers (CSFs) with low (

Angiotensin II (ANG II) raises blood pressure partly by stimulating tubular Na+ reabsorption. The effects of ANG II on tubular Na+ transporters (i.e., channels, pumps, cotransporters, and exchangers) vary between short-term and long-term exposure. To better understand the physiological impact, we used a computational model of transport along the rat nephron to predict the effects of short- and long-term ANG II-induced transporter activation on Na+ and K+ reabsorption/secretion, and to compare measured and calculated excretion rates. Three days of ANG II infusion at 200 ng·kg−1·min−1 is nonpressor, yet stimulates transporter accumulation. The increase in abundance of Na+/H+ exchanger 3 (NHE3) or activated Na+-K+-2Cl− cotransporter-2 (NKCC2-P) predicted significant reductions in urinary Na+ excretion, yet there was no observed change in urine Na+. The lack of antinatriuresis, despite Na+ transporter accumulation, was supported by Li+ and creatinine clearance measurements, leading to the conclusion that 3-day nonpressor ANG II increases transporter abundance without proportional activation. Fourteen days of ANG II infusion at 400 ng·kg−1·min−1 raises blood pressure and increases Na+ transporter abundance along the distal nephron; proximal tubule and medullary loop transporters are decreased and urine Na+ and volume output are increased, evidence for pressure natriuresis. Simulations indicate that decreases in NHE3 and NKCC2-P contribute significantly to reducing Na+ reabsorption along the nephron and to pressure natriuresis. Our results also suggest that differential regulation of medullary (decrease) and cortical (increase) NKCC2-P is important to preserve K+ while minimizing Na+ retention during ANG II infusion. Lastly, our model indicates that accumulation of active Na+-Cl− cotransporter counteracts epithelial Na+ channel-induced urinary K+ loss.

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.

Circadian rhythms govern physiological functions and are important for overall health. The molecular circadian clock comprises several transcription factors that mediate circadian control of physiological function, in part, by regulating gene expression in a tissue-specific manner. These connections are well established, but the underlying mechanisms are incompletely understood. The overall goal of this study was to examine the connection among the circadian clock protein Period 1 (Per1), epithelial Na+ channel (ENaC), and blood pressure (BP) using a multipronged approach. Using global Per1 knockout mice on a 129/sv background in combination with a high-salt diet plus mineralocorticoid treatment, we demonstrated that loss of Per1 in this setting is associated with protection from hypertension. Next, we used the ENaC inhibitor benzamil to demonstrate a role for ENaC in BP regulation and urinary Na+ excretion in 129/sv mice. We targeted Per1 indirectly using pharmacological inhibition of Per1 nuclear entry in vivo to demonstrate altered expression of known Per1 target genes as well as a BP-lowering effect in 129/sv mice. Finally, we directly inhibited Per1 via genetic knockdown in amphibian distal nephron cells to demonstrate, for the first time, that reduced Per1 expression is associated with decreased ENaC activity at the single channel level.

Stimulation of metabotropic Gq-coupled purinergic P2Y2receptors decreases activity of the epithelial Na+channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2receptor signaling disruption in the P2Y2receptor knockout mouse are decreased Na+excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2receptor, we test the sufficiency of targeted stimulation of Gqsignaling in principal cells of the distal nephron and P2Y2receptors to increase UNaV. Selective stimulation of the P2Y2receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2receptor and Gqsignaling in principal cells is sufficient to promote renal salt excretion.

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGCis controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa; and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague-Dawley (8) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSFis an indirect measurement of PGC. Maximal TGF response at 40 nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats, indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker benzamil (1 μM/l), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.

A proper morphogenesis of the renal microvasculature is crucial not only for fulfilling the renal function but also to slow down the progression of chronic kidney disease in adulthood. However, the current description of the developing microvasculature is incomplete. The present study investigated the morphogenesis and volume densities of the renal microvasculature using computer-assisted tubular tracing, immunohistochemistry for CD34, and unbiased stereology. The earliest glomerular capillaries were observed at the lower cleft of the S-shaped nephrons, as simple loops connecting the afferent and efferent arterioles. In parallel with this, the peritubular capillaries were established. Noticeably, from early nephrogenesis on, the efferent arterioles of the early-formed glomeruli ran in close proximity to their own thick ascending limbs. In addition, the ascending vasa recta arising from the arcuate or interlobular veins also ran in close proximity to the thick descending limb. Thus, the tubules and vessels formed the typical countercurrent relation in the medulla. No loop bends were observed between descending and ascending vasa recta. The volume density of the cortical and medullary peritubular capillary increased 3.3- and 2.6-fold, respectively, from 2.34 (0.13) and 7.03 (0.09)% [means (SD)] at embryonic day 14.5 (E14.5) to 7.71 (0.44) and 18.27 (1.17)% at postnatal day 40 (P40). In contrast, the volume density of glomeruli changed only slightly during kidney development, from 4.61 (0.47)% at E14.5 to 6.07 (0.2)% at P7 to 4.19 (0.47)% at P40. These results reflect that the growth and formation of the renal microvasculature closely correspond to functional development of the tubules.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors enhance urinary glucose, Na+and fluid excretion, and lower hyperglycemia in diabetes by targeting Na+and glucose reabsorption along the proximal convoluted tubule. A goal of this study was to predict the effects of SGLT2 inhibitors in diabetic and nondiabetic patients with chronic kidney disease. To that end, we employed computational rat kidney models to explore how SGLT2 inhibition affects renal solute transport and metabolism when nephron populations are normal or reduced. Model simulations suggested that in a nondiabetic rat, acute and chronic SGLT2 inhibition induces glucosuria, diuresis, natriuresis, and kaliuresis. Those effects were stronger with chronic SGLT2 inhibition (due to SGLT1 downregulation) and tempered by nephron loss. In a diabetic rat with normal nephron number, acute SGLT2 inhibition similarly elevated urine fluid, Na+, and K+excretion, whereas the urinary excretory effects of chronic SGLT2 inhibition were attenuated in proportion to its plasma glucose level lowering effect. Nephron loss in a diabetic kidney was predicted to lower the glucosuric and blood glucose-reducing effect of chronic SGLT2 inhibition, but due to the high luminal glucose delivery in the remaining hyperfiltering nephrons, nephron loss enhanced proximal tubular paracellular Na+secretion, thereby augmenting the natriuretic, diuretic, and kaliuretic effects. A proposed shift in oxygen-consuming active transport to the outer medulla, which may simulate systemic hypoxia and enhance erythropoiesis, was also preserved with nephron loss. These effects may contribute to the protective effects of SGLT2 inhibitors on blood pressure and heart failure observed in diabetic patients with chronic kidney diseases.

Reversible posttranslational modification of proteins is a critically important process in physiological regulation in all tissues, including the kidney. Lysine acetylation occurs in all organisms, including prokaryotes, and is regulated by a balance between the lysine acetyltransferases (adding an acetyl group to the ε-amino group of a lysine) and deacetylases (removing it). The kidney is an organ rich with acetylated lysines, which map to >2,000 unique histone and nonhistone proteins. However, the functional significance of these modifications remains to be discovered. Here, we have compiled gene lists of the acetyltransferases and deacetylases in the mammalian genomes and mapped their mRNA expression along the renal tubule. These lists will be useful for generating targeted approaches to test the physiological or pathophysiological significance of lysine acetylation changes in the kidney.

We assessed effects of acute volume reductions induced by administration of diuretics in rats. Direct block of Na+ transport produced changes in urinary electrolyte excretion. Adaptations to these effects appeared as alterations in the expression of protein for the distal nephron Na+ transporters NCC and ENaC. Two hours after a single injection of furosemide (6 mg/kg) or hydrochlorothiazide (HCTZ; 30 mg/kg) Na+ and K+ excretion increased but no changes in the content of activated forms of NCC (phosphorylated on residue T53) or ENaC (cleaved γ-subunit) were detected. In contrast, amiloride (0.6 mg/kg) evoked a similar natriuresis that coincided with decreased pT53NCC and increased cleaved γENaC. Alterations in posttranslational membrane protein processing correlated with an increase in plasma K+ of 0.6–0.8 mM. Decreased pT53NCC occurred within 1 h after amiloride injection, whereas changes in γENaC were slower and were blocked by the mineralocorticoid receptor antagonist spironolactone. Increased γENaC cleavage correlated with elevation of the surface expression of the subunit as assessed by in situ biotinylation. Na depletion induced by 2 h of furosemide or HCTZ treatment increases total NCC expression without affecting ENaC protein. However, restriction of Na intake for 10 h (during the day) or 18 h (overnight) increased the abundance of both total NCC and of cleaved α- and γENaC. We conclude that the kidneys respond acutely to hyperkalemic challenges by decreasing the activity of NCC while increasing that of ENaC. They respond to hypovolemia more slowly, increasing Na+ reabsorptive capacities of both of these transporters.

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

The paracellular pathways in renal tubular epithelia such as the proximal tubules, which reabsorb the largest fraction of filtered solutes and water and are leaky epithelia, are important routes for transepithelial transport of solutes and water. Movement occurs passively via an extracellular route through the tight junction between cells. The characteristics of paracellular transport vary among different nephron segments with leaky or tighter epithelia. Claudins expressed at tight junctions form pores and barriers for paracellular transport. Claudins are from a multigene family, comprising at least 27 members in mammals. Multiple claudins are expressed at tight junctions of individual nephron segments in a nephron segment-specific manner. Over the last decade, there have been advances in our understanding of the structure and functions of claudins. This paper is a review of our current knowledge of claudins, with special emphasis on their physiological roles in proximal tubule paracellular solute and water transport.

MRI can provide excellent detail of renal structure and function. Recently, novel MR contrast mechanisms and imaging tools have been developed to evaluate microscopic kidney structures including the tubules and glomeruli. Quantitative MRI can assess local tubular function and is able to determine the concentrating mechanism of the kidney noninvasively in real time. Measuring single nephron function is now a near possibility. In parallel to advancing imaging techniques for kidney microstructure is a need to carefully understand the relationship between the local source of MRI contrast and the underlying physiological change. The development of these imaging markers can impact the accurate diagnosis and treatment of kidney disease. This study reviews the novel tools to examine kidney microstructure and local function and demonstrates the application of these methods in renal pathophysiology.

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

The kidney continues to mature postnatally, with significant elongation of nephron tubules and collecting ducts to maintain fluid/electrolyte homeostasis. The aim of this project was to develop methodology to estimate lengths of specific segments of nephron tubules and collecting ducts in the CD-1 mouse kidney using a combination of immunohistochemistry and design-based stereology (vertical uniform random sections with cycloid arc test system). Lengths of tubules were determined at postnatal day 21 (P21) and 2 and 12 mo of age and also in mice fed a high-salt diet throughout adulthood. Immunohistochemistry was performed to identify individual tubule segments [aquaporin-1, proximal tubules (PT) and thin descending limbs of Henle (TDLH); uromodulin, distal tubules (DT); aquaporin-2, collecting ducts (CD)]. All tubular segments increased significantly in length between P21 and 2 mo of age (PT, 602% increase; DT, 200% increase; TDLH, 35% increase; CD, 53% increase). However, between 2 and 12 mo, a significant increase in length was only observed for PT (76% increase in length). At 12 mo of age, kidneys of mice on a high-salt diet demonstrated a 27% greater length of the TDLH, but no significant change in length was detected for PT, DT, and CD compared with the normal-salt group. Our study demonstrates an efficient method of estimating lengths of specific segments of the renal tubular system. This technique can be applied to examine structure of the renal tubules in combination with the number of glomeruli in the kidney in models of altered renal phenotype.

Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress, and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 in the generation of H2O2and O2·−in the renal medullary thick ascending limb of Henle (mTAL) of SS rats in response to increasing luminal flow (from 5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the mTAL of SS rats. Responses of SS rats were compared with those of SS rats with knockout of Nox4 (SSNox4−/−) or functional mutation of p67phox (SSp67phox−/−). Nox4 was the dominant source of increased intracellular H2O2production in response to increased luminal flow as determined using the fluorescent dye peroxyfluor 6-AM (PF6-AM). The rate of mitochondrial H2O2production [as determined by mitochondria peroxy yellow 1 (mitoPY1)] was also significantly reduced in SSNox4−/−compared with SS rats, but not in SSp67phox−/−rats. In contrast, intracellular superoxide (O2·−) production (the ratio of ethidium to dihydroethidium) in the mTAL of SSNox4−/−rats was nearly identical to that of SS rats in response to luminal flow, indicating that Nox4 made no measurable contribution. mTAL O2·−production was reduced in SSp67phox−/−compared with SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases in intracellular and mitochondrial H2O2, which are dependent on the presence of Nox4, and that p67phox/Nox2 accounts solely for increases in O2·−production.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na+-K+-ATPase (EC 3.6.3.9), secondary active Na+-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na+-K+-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1–OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

Diuretics acting on specific nephron segments to inhibit Na+ reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+ complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na+-K+-2Cl− cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na+ channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+ excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K+ secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca2+-activated K+ channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target.

Mammalian class IX myosin Myo9a is a single-headed, actin-dependent motor protein with Rho GTPase-activating protein activity that negatively regulates Rho GTPase signaling. Myo9a is abundantly expressed in ciliated epithelial cells of several organs. In mice, genetic deletion of Myo9a leads to the formation of hydrocephalus. Whether Myo9a also has essential functions in the epithelia of other organs of the body has not been explored. In the present study, we report that Myo9a-deficient mice develop bilateral renal disease, characterized by dilation of proximal tubules, calyceal dilation, and thinning of the parenchyma and fibrosis. These structural changes are accompanied by polyuria (with normal vasopressin levels) and low-molecular-weight proteinuria. Immunohistochemistry revealed that Myo9a is localized to the circumferential F-actin belt of proximal tubule cells. In kidneys lacking Myo9a, the multiligand binding receptor megalin and its ligand albumin accumulated at the luminal surface of Myo9a-deficient proximal tubular cells, suggesting that endocytosis is dysregulated. In addition, we found, surprisingly, that levels of murine diaphanous-related formin-1, a Rho effector, were decreased in Myo9a-deficient kidneys as well as in Myo9a knockdown LLC-PK1 cells. In summary, deletion of the Rho GTPase-activating protein Myo9a in mice causes proximal tubular dilation and fibrosis, and we speculate that downregulation of murine diaphanous-related formin-1 and impaired protein reabsorption contribute to the pathophysiology.

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane.

Arginine vasopressin (AVP) is released from the posterior pituitary gland during states of hyperosmolality or hypovolemia. AVP is a peptide hormone, with antidiuretic and antinatriuretic properties. It allows the kidneys to increase body water retention predominantly by increasing the cell surface expression of aquaporin water channels in the collecting duct alongside increasing the osmotic driving forces for water reabsorption. The antinatriuretic effects of AVP are mediated by the regulation of sodium transport throughout the distal nephron, from the thick ascending limb through to the collecting duct, which in turn partially facilitates osmotic movement of water. In this review, we will discuss the regulatory role of AVP in sodium transport and summarize the effects of AVP on various molecular targets, including the sodium-potassium-chloride cotransporter NKCC2, the thiazide-sensitive sodium-chloride cotransporter NCC, and the epithelial sodium channel ENaC.

The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm2) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.

Mathematical models of the proximal tubule (PT), loop of Henle (LOH), and distal nephron have been combined to simulate transport by rat renal tubules. The ensemble is composed of 24,000 superficial (SF) nephrons and 12,000 juxtamedullary (JM) nephrons in 5 classes (according to LOH length); all coalesce into 7,200 connecting tubules (CNT). Medullary interstitial solute concentrations are specified. The model equations require that each nephron glomerular filtration rate (GFR) satisfies a tubuloglomerular feedback (TGF) relationship, and each initial hydrostatic pressure yields a common CNT pressure; that common CNT pressure is determined from an overall distal hydraulic resistance to flow. By virtue of the greater GFR for JM nephrons, fluid delivery to SF and JM tubules is comparable. Glucose reabsorption is restricted to the PT, cotransported with one Na in the convoluted tubule (SGLT2), and two Na in the straight tubule (SGLT1). Increasing ambient glucose from 5 to 10 mM increases proximal Na reabsorption and decreases distal delivery. This is mitigated by a TGF-mediated increase in GFR, and may thus be an etiology for TGF-mediated glomerular hyperfiltration. With SGLT2 inhibition by 95%, the model predicts that under normoglycemic conditions about 60% of filtered glucose will still be reabsorbed, so that profound glycosuria is not to be expected. Compared with glucose-driven osmotic diuresis, SGLT2 inhibition provokes greater natriuresis. When hyperglycemia is superimposed on SGLT2 inhibition, the model suggests that natriuresis may be severe, reflecting synergy of a proximal diuretic and osmotic diuresis. In sum, the model captures TGF-mediated diabetic hyperfiltration and predicts glomerular protection with SGLT2 inhibition.

Insulin and IGF-1 are recognized as powerful regulators of the epithelial Na+channel (ENaC) in the aldosterone-sensitive distal nephron. As previously described, these hormones both acutely increase ENaC activity in freshly isolated split open tubules and cultured principal cortical collecting duct cells. The present study was aimed at differentiating the effects of insulin and IGF-1 on Na+transport in immortalized mpkCCDcl4cells and defining their interrelations. We have shown that both insulin and IGF-1 applied basolaterally, but not apically, enhanced transepithelial Na+transport in the mpkCCDcl4cell line with EC50values of 8.8 and 14.5 nM, respectively. Insulin treatment evoked phosphorylation of both insulin and IGF-1 receptors, whereas the effects of IGF-1 were more profound on its own receptor rather than the insulin receptor. AG-1024 and PPP, inhibitors of IGF-1 and insulin receptor tyrosine kinase activity, diminished insulin- and IGF-1-stimulated Na+transport in mpkCCDcl4cells. The effects of insulin and IGF-1 on ENaC-mediated currents were found to be additive, with insulin likely stimulating both IGF-1 and insulin receptors. We hypothesize that insulin activates IGF-1 receptors in addition to its own receptors, making the effects of these hormones interconnected.

Synchronization of tubuloglomerular feedback (TGF) dynamics in nephrons that share a cortical radial artery is well known. It is less clear whether synchronization extends beyond a single cortical radial artery or whether it extends to the myogenic response (MR). We used LSCI to examine cortical perfusion dynamics in isoflurane-anesthetized, male Long-Evans rats. Inhibition of nitric oxide synthases by Nω-nitro-l-arginine methyl ester (l-NAME) was used to alter perfusion dynamics. Phase coherence (PC) was determined between all possible pixel pairs in either the MR or TGF band (0.09–0.3 and 0.015–0.06 Hz, respectively). The field of view (≈4 × 5 mm) was segmented into synchronized clusters based on mutual PC. During the control period, the field of view was often contained within one cluster for both MR and TGF. PC was moderate for TGF and modest for MR, although significant in both. In both MR and TGF, PC exhibited little spatial variation. After l-NAME, the number of clusters increased in both MR and TGF. MR clusters became more strongly synchronized while TGF clusters showed small highly coupled, high-PC regions that were coupled with low PC to the remainder of the cluster. Graph theory analysis probed modularity of synchronization. It confirmed weak synchronization of MR during control that probably was not physiologically relevant. It confirmed extensive and long-distance synchronization of TGF during control and showed increased modularity, albeit with larger modules seen in MR than in TGF after l-NAME. The results show widespread synchronization of MR and TGF that is differentially affected by nitric oxide.

Long-standing experimental evidence suggests that epithelial cells in the renal tubule are able to sense osmotic and pressure gradients caused by alterations in ultrafiltrate flow by elevating intracellular Ca2+concentration. These responses are viewed as critical regulators of a variety of processes ranging from transport of water and solutes to cellular growth and differentiation. A loss in the ability to sense mechanical stimuli has been implicated in numerous pathologies associated with systemic imbalance of electrolytes and to the development of polycystic kidney disease. The molecular mechanisms conferring mechanosensitive properties to epithelial tubular cells involve activation of transient receptor potential (TRP) channels, such as TRPV4, allowing direct Ca2+influx to increase intracellular Ca2+concentration. In this review, we critically analyze the current evidence about signaling determinants of TRPV4 activation by luminal flow in the distal nephron and discuss how dysfunction of this mechanism contributes to the progression of polycystic kidney disease. We also review the physiological relevance of TRPV4-based mechanosensitivity in controlling flow-dependent K+secretion in the distal renal tubule.

DNA label-retention, or retention of a thymidine analog, is a characteristic of slow cycling cells and has been used to identify stem cells in several organ systems. Recent findings have demonstrated inconsistent localization of label-retaining cells (LRCs) in the kidney. Differences in the dose and timing of administration of deoxyuridine, the length of the chase period, and the species of animal used have made understanding the distinctions between these findings difficult. In the present studies, we utilized a dual loading scheme in the same animal to demonstrate that the cells labeled at different ages identified independent populations of LRC that distributed globally in an anti-parallel manner in the kidney. Loading with a DNA label in neonates identified LRC more often in the papilla, while administering the DNA label in adult mice identified LRC prominently in the cortex and the outer medulla. Furthermore, the tissue compartment distribution (epithelial-endothelial-interstitial) as well as the specific distribution within the nephron epithelia differed for these populations. These findings highlighted the complexity of the dynamics of cell proliferation in the kidney throughout the postnatal and adult period and call attention to the confusion associated with the term “label-retaining cells” for different timings of the loading and chase periods. This study indicated that the results of previous studies should be viewed as nonoverlapping and that further studies are needed to ascertain the role of each of these populations in the steady-state maintenance and injury recovery of the kidney.

During kidney development, the vasculature develops via both angiogenesis (branching from major vessels) and vasculogenesis (de novo vessel formation). The formation and perfusion of renal blood vessels are vastly understudied. In the present study, we investigated the regulatory role of renal blood flow and O2 concentration on nephron progenitor differentiation during ontogeny. To elucidate the presence of blood flow, ultrasound-guided intracardiac microinjection was performed, and FITC-tagged tomato lectin was perfused through the embryo. Kidneys were costained for the vasculature, ureteric epithelium, nephron progenitors, and nephron structures. We also analyzed nephron differentiation in normoxia compared with hypoxia. At embryonic day 13.5 (E13.5), the major vascular branches were perfused; however, smaller-caliber peripheral vessels remained unperfused. By E15.5, peripheral vessels started to be perfused as well as glomeruli. While the interior kidney vessels were perfused, the peripheral vessels (nephrogenic zone) remained unperfused. Directly adjacent and internal to the nephrogenic zone, we found differentiated nephron structures surrounded and infiltrated by perfused vessels. Furthermore, we determined that at low O2 concentration, little nephron progenitor differentiation was observed; at higher O2 concentrations, more differentiation of the nephron progenitors was induced. The formation of the developing renal vessels occurs before the onset of blood flow. Furthermore, renal blood flow and oxygenation are critical for nephron progenitor differentiation.

Adenylyl cyclases (AC) catalyze formation of cAMP, a critical component of G protein-coupled receptor signaling. So far, nine distinct membrane-bound AC isoforms (AC1-9) and one soluble AC (sAC) have been identified and, except for AC8, all of them are expressed in the kidney. While the role of ACs in renal cAMP formation is well established, we are just beginning to understand the function of individual AC isoforms, particularly with regard to hormonal regulation of transporter and channel phosphorylation, membrane abundance, and trafficking. This review focuses on the role of different AC isoforms in regulating renal water and electrolyte transport in health as well as potential pathological implications of disordered AC isoform function. In particular, we focus on modulation of transporter and channel abundance, activity, and phosphorylation, with an emphasis on studies employing genetically modified animals. As will be described, it is now evident that specific AC isoforms can exert unique effects in the kidney that may have important implications in our understanding of normal physiology as well as disease pathogenesis.

This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. Approximately 4,500 2.5-μm-thick serial sections from the renal surface to the papillary tip were obtained from each of 3 kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e., the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons, a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal “straight” tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli; i.e., the deeper the glomerulus, the deeper the bend. Each CD drained approximately three to six nephrons with a different pattern than previously established in mice. This information will provide a basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.

We present a lumped-nephron model that explicitly represents the main features of the underlying physiology, incorporating the major hormonal regulatory effects on both tubular and vascular function, and that accurately simulates hormonal regulation of renal salt and water excretion. This is the first model to explicitly couple glomerulovascular and medullary dynamics, and it is much more detailed in structure than existing whole organ models and renal portions of multiorgan models. In contrast to previous medullary models, which have only considered the antidiuretic state, our model is able to regulate water and sodium excretion over a variety of experimental conditions in good agreement with data from experimental studies of the rat. Since the properties of the vasculature and epithelia are explicitly represented, they can be altered to simulate pathophysiological conditions and pharmacological interventions. The model serves as an appropriate starting point for simulations of physiological, pathophysiological, and pharmacological renal conditions and for exploring the relationship between the extrarenal environment and renal excretory function in physiological and pathophysiological contexts.

Renal intercalated cells mediate the secretion or absorption of Cl−and OH−/H+equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl−absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl−transporter(s) are targeted by these diuretics is debated. While epithelial Na+channel (ENaC) does not transport Cl−, it modulates Cl−transport probably by generating a lumen-negative voltage, which drives Cl−flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl−secretion via a type A intercalated cells. During ENaC blockade, Cl−is taken up across the basolateral membrane through the Na+-K+−2Cl−cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl−channel or an electrogenic exchanger). The mechanism of this apical Cl−secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl−absorption mediated by a Na+-dependent Cl−/HCO3−exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl−absorption varies between studies and probably depends on the treatment model employed. Cl−absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na+-independent Cl−/HCO3−exchanger pendrin. In the absence of pendrin [ Slc26a4(−/−)or pendrin null mice], aldosterone-stimulated Cl−absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3−. This review summarizes mechanisms of Cl−transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

A serine-threonine protein kinase, WNK4, reduces Na+reabsorption and K+secretion in the distal convoluted tubule by reducing trafficking of the thiazide-sensitive Na-Cl cotransporter to and enhancing renal outer medullary potassium channel retrieval from the apical membrane. Epithelial sodium channels (ENaC) in the distal nephron also play a role in regulating Na+reabsorption and are also regulated by WNK4, but the mechanism is unclear. In A6 distal nephron cells, transepithelial current measurement and single channel recording show that WNK4 inhibits ENaC activity. Analysis of the number of channel per patch shows that WNK4 reduces channel number but has no effect on channel open probability. Western blots of apical and total ENaC provide additional evidence that WNK4 reduces apical as well as total ENaC expression. WNK4 enhances ENaC internalization independent of Nedd4-2-mediated ENaC ubiquitination. WNK4 also reduced the amount of ENaC available for recycling but has no effect on the rate of transepithelial current increase to forskolin. In contrast, Nedd4-2 not only reduced ENaC in the recycling pool but also decreased the rate of increase of current after forskolin. WNK4 associates with wild-type as well as Liddle's mutated ENaC, and WNK4 reduces both wild-type and mutated ENaC expressed in HEK293 cells.

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-β4 in acid/base-transporting intercalated cells (IC), we determined whether BK-β4 knockout mice (β4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-β4 expression increased (Western blot). When WT and β4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and β4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with β4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and β4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in β-IC, comparing WT and β4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in β4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of β4KO. When WT on LNaHK were given NH4Cl in the drinking water, K excretion was reduced to the magnitude of β4KO on LNaHK. These results show that WT, but not β4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/β4-mediated K secretion is promoted by bicarbonaturia.

Preterm neonates are commonly exposed postnatally to pharmacological treatments for a patent ductus arteriosus. Exposure of the developing kidney to nephrotoxic medications may adversely impact renal development. This study aimed to determine the effect of early postnatal ibuprofen treatment, both alone and in combination with a nitric oxide synthase inhibitor (NOSi), on renal development and morphology. Baboon neonates were delivered prematurely at 125-day (125d) gestation (term = 185d) and were euthanized at birth or postnatal day 6. Neonates were divided into four groups: 125d gestational controls ( n = 8), Untreated ( n = 8), Ibuprofen ( n = 6), and ibuprofen (Ibu)+NOSi ( n = 4). Animals in the Ibuprofen and Ibu+NOSi groups received five doses of ibuprofen, with the Ibuprofen+NOSi animals additionally administered a NOS inhibitor ( NG-monomethyl-l-arginine). There was no difference among groups in body weight, kidney weight, or glomerular generation number. Nephrogenic zone width was significantly reduced in the Ibuprofen group (123.5 ± 7.4 μm) compared with the 125d gestational control (176.1 ± 6.9 μm) and Untreated animals (169.7 ± 78.8 μm). In the Ibu+NOSi group, nephrogenic zone width averaged 152.7 ± 3.9 μm, which was not significantly different from any other group. Morphologically abnormal glomeruli were present at a range of 0.0–22.9% in the Untreated group, 0.0–6.1% in the Ibuprofen group, and 0.0–1.4% in the Ibu+NOSi group. In conclusion, early postnatal ibuprofen exposure is associated with a reduced nephrogenic zone width, which may suggest the early cessation of nephrogenesis following treatment. Ultimately, this may impact the number of nephrons formed in the preterm kidney.

Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE2and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE2clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE2, and 15-PGDH in the renal cortex. Tissue PGE2concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE2concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE2concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE2clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE2clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE2in the kidney.

There is increasing evidence that proteins in tubular fluid are “nephrotoxic.” In vivo it is difficult to study protein loading of tubular epithelial cells in isolation, i.e., without concomitant glomerular damage or changes of renal hemodynamics, etc. Recently, a unique amphibian model has been described which takes advantage of the special anatomy of the amphibian kidney in which a subset of nephrons drains the peritoneal cavity (open nephrons) so that intraperitoneal injection of protein selectively causes protein storage in and peritubular fibrosis around open but not around closed tubules. There is an ongoing debate as to what degree albumin per se is nephrotoxic and whether modification of albumin alters its nephrotoxicity. We tested the hypothesis that carbamylation and glycation render albumin more nephrotoxic compared with native albumin and alternative albumin modifications, e.g., lipid oxidation and lipid depletion. Preparations of native and modified albumin were injected into the axolotl peritoneum. The kidneys were retrieved after 10 days and studied by light microscopy as well as by immunohistochemistry [transforming growth factor (TGF)-β, PDGF, NF-κB, collagen I and IV, RAGE], nonradioactive in situ hybridization, and Western blotting. Two investigators unaware of the animal groups evaluated and scored renal histology. Compared with unmodified albumin, glycated and carbamylated albumin caused more pronounced protein storage. After no more than 10 days, selective peritubular fibrosis was seen around nephrons draining the peritoneal cavity (open nephrons), but not around closed nephrons. Additionally, more intense expression of RAGE, NF-κB, as well as PDGF, TGF-β, EGF, ET-1, and others was noted by histochemistry and confirmed by RT-PCR for fibronectin and TGF-β as well as nonradioactive in situ hybridization for TGF-β and fibronectin. The data indicate that carbamylation and glycation increase the capacity of albumin to cause tubular cell damage and peritubular fibrosis.

We recently standardized a model (LLact) of severe chronic kidney disease based on impaired nephrogenesis by suppression of angiotensin II activity during lactation (Machado FG, Poppi EP, Fanelli C, Malheiros DM, Zatz R, Fujihara CK. Am J Physiol Renal Physiol 294: F1345–F1353, 2008). In this new study of the LLactmodel, we sought to gain further insight into renal injury mechanisms associated with this model and to verify whether the renoprotection obtained with the association of the angiotensin II receptor blocker losartan (L) and hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, would provide similar renoprotection. Twenty Munich-Wistar dams, each nursing six pups, were divided into control, untreated, and LLactgroups, given losartan (L; 250 mg·kg−1·day−1) until weaning. The male LLactoffspring remained untreated until 7 mo of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (LLactPre), and followed no further. The remaining rats were then divided among groups LLact+V, untreated; LLact+L, given L (50 mg·kg−1·day−1) now as a therapy; LLact+H, given H (6 mg·kg−1·day−1); and LLact+LH, given L and H. All parameters were reassessed 3 mo later in these groups and in age-matched controls. At this time, LLactrats exhibited hypertension, severe albuminuria, glomerular damage, marked interstitial expansion/inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. L monotherapy normalized albuminuria and prevented hypertension and the progression of renal injury, inflammation, and myofibroblast infiltration. In contrast to the remnant model, the LH combination promoted only slight additional renoprotection, perhaps because of a limited tendency to retain sodium in LLactrats.

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD ( P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.