The Convoluted Tubules of the Nephron Must Be Considered Elliptical, and Not Circular, in Stereological Studies of the Kidney.

INTRODUCTION: The diameter and area of the proximal convoluted tubule (PCT) and the distal convoluted tubule (DCT) are of the main parameters analyzed in stereological studies of the kidney. However, there is no consensus about if the PCT and DCT should be considered circular or elliptical in shape. OBJECTIVE: To analyze if there are significant differences in the diameter and area of the PCT and DCT, depending on whether they are considered circular or elliptical. METHODS: Paraffin-embedded sections of kidneys from CD1 mice were stained with hematoxylin and eosin and examined using a light microscope. Images were captured using a camera linked to image analysis software. A short diameter (d) and a long diameter (D) were measured in both PCT and DCT. A small circular area (SCA), a large circular area (LCA), and an elliptical area (EA) were calculated with mathematical formulas that incorporate d and D values, while a program area (PA) was provided by the software. RESULTS: There was a significant difference between d and D in both PCT (F = 1.354, Sig = 0.000) and DCT (F = 4.989, Sig = 0.000). Also, there were significant differences in the tubular areas in both PCT (F = 34.843, Sig = 0.000) and DCT (F = 22.390, Sig = 0.000); circular areas were different from elliptical areas (SCA and LCA vs. EA and PA). CONCLUSION: The convoluted tubules of the nephron must not be considered circular, but rather elliptical; care should be taken every time the tubules are analyzed in stereological studies of the kidney, especially when evaluating their diameters and areas.

Mapping nephron mass in vivo using positron emission tomography.

Nephron number varies widely in humans. A low nephron endowment at birth or a loss of functioning nephrons is strongly linked to increased susceptibility to chronic kidney disease. In this work, we developed a contrast agent, radiolabeled cationic ferritin (RadioCF), to map functioning glomeruli in vivo in the kidney using positron emission tomography (PET). PET radiotracers can be detected in trace doses (<30 nmol), making them useful for rapid clinical translation. RadioCF is formed from cationic ferritin (CF) and with a radioisotope, Cu-64, incorporated into the ferritin core. We showed that RadioCF binds specifically to kidney glomeruli after intravenous injection in mice, whereas radiolabeled noncationic ferritin (RadioNF) and free Cu-64 do not. We then showed that RadioCF-PET can distinguish kidneys in healthy wild-type (WT) mice from kidneys in mice with oligosyndactylism (Os/+), a model of congenital hypoplasia and low nephron mass. The average standardized uptake value (SUV) measured by PET 90 min after injection was 21% higher in WT mice than in Os/+ mice, consistent with the higher glomerular density in WT mice. The difference in peak SUV from SUV at 90 min correlated with glomerular density in male mice from both WT and Os/+ cohorts (R2 = 0.98). Finally, we used RadioCF-PET to map functioning glomeruli in a donated human kidney. SUV within the kidney correlated with glomerular number (R2= 0.78) measured by CF-enhanced magnetic resonance imaging in the same locations. This work suggests that RadioCF-PET appears to accurately detect nephron mass and has the potential for clinical translation.

Kidney functional morphology variations between spring and winter in the Saharan male lizard Uromastyx acanthinura (Sauria, Agamidae), with special reference to body water economy.

To better understand the adaptive mechanisms in Uromastyx acanthinura to the seasonal variations in the arid environment, the present study aimed to explore the kidney functional morphology involved in body water economy. These investigations were carried out by the histological, histochemical and immuno-histochemical methods using conventional light microscopy. The glomeruli number is estimated at 2000 per kidney. The glomeruli size is rather small and decreases significantly in winter. Interestingly, the proximal convoluted tubule (PCT) is long and divided into two different segments which is one of the particularities of this species. Both of the distal convoluted tubule (DCT), connecting tubule (CnT) and collecting duct (CD) epithelium contains mucous cells. The nature and intensity of these mucous secretions vary according to seasons. The evident hypertrophy of the secondary collecting duct (SCD) and tertiary collecting duct (TCD) epithelium is related to the high secretory activity during spring, corresponding to the sexual segment of kidney (SSK). Labeling with anti α-smooth muscle actin-1 showed a thick layer of mucularis surrounded the entire CD. Also, the mesangium of glomeruli contains myofibroblasts. All these renal structural characteristics involved in body water economy may be considered as an adaptive mechanisms of U. acanthinura to resist to dehydration and cope with seasonal variations in the arid environment.

Facilitated NaCl Uptake in the Highly Developed Bundle of the Nephron in Japanese Red Stingray Hemitrygon akajei Revealed by Comparative Anatomy and Molecular Mapping.

Batoidea (rays and skates) is a monophyletic subgroup of elasmobranchs that diverged from the common ancestor with Selachii (sharks) about 270 Mya. A larger number of batoids can adapt to low-salinity environments, in contrast to sharks, which are mostly stenohaline marine species. Among osmoregulatory organs of elasmobranchs, the kidney is known to be dedicated to urea retention in ureosmotic cartilaginous fishes. However, we know little regarding urea reabsorbing mechanisms in the kidney of batoids. Here, we performed physiological and histological investigations on the nephrons in the red stingray (Hemitrygon akajei) and two shark species. We found that the urine/plasma ratios of salt and urea concentrations in the stingray are significantly lower than those in cloudy catshark (Scyliorhinus torazame) under natural seawater, indicating that the kidney of stingray more strongly reabsorbs these osmolytes. By comparing the three-dimensional images of nephrons between stingray and banded houndshark (Triakis scyllium), we showed that the tubular bundle of stingray has a more compact configuration. In the compact tubular bundle of stingray kidney, the distal diluting tubule was highly developed and frequently coiled around the proximal and collecting tubules. Furthermore, co-expression of NKAα1 (Na+/K +-ATPase) and NKCC2 (Na+- K+-2Cl- cotransporter 2) mRNAs was prominent in the coiled diluting segment. These findings imply that NaCl reabsorption is greatly facilitated in the stingray kidney, resulting in a higher reabsorption rate of urea. Lowering the loss of osmolytes in the glomerular filtrate is likely favorable to the adaptability of batoids to a wide range of environmental salinity.

A practical guide to the stereological assessment of glomerular number, size, and cellular composition.

The evaluation of a range of measures in the kidneys, such as developmental stage, rate and success, injury, and disease processes, relies on obtaining information on the three-dimensional structure of the renal corpuscles, and in particular the glomerular capillary tufts. To do this in the most accurate, comprehensive, and unbiased manner depends on a knowledge of stereological methods. In this article, we provide a practical guide for researchers on how to quantitate a number of structures in the kidneys, including the estimation of total glomerular number, glomerular capillary length and filtration surface area, and the cellular composition of individual glomeruli. Guidance is also provided on how to apply these methods to kidneys at different sizes and levels of maturity.

In Search of Imprints Left by the Impairment of Nephrogenesis.

Clinical aspects dealing with the impairment of nephrogenesis in preterm and low birth weight babies were intensely researched. In this context it was shown that quite different noxae can harm nephron formation, and that the morphological damage in the fetal kidney is rather complex. Some pathological findings show that the impairment leads to changes in developing glomeruli that are restricted to the maturation zone of the outer cortex in the fetal human kidney. Other data show also imprints on the stages of nephron anlage including the niche, the pretubular aggregate, the renal vesicle, and comma- and S-shaped bodies located in the overlying nephrogenic zone of the rodent and human kidneys. During our investigations it was noticed that the stages of nephron anlage in the fetal human kidney during the phase of late gestation have not been described in detail. To contribute, these stages were recorded along with corresponding images. The initial nephron formation in the rodent kidney served as a reference. Finally, the known imprints left by the impairment in both specimens were listed and discussed. In sum, the relatively paucity of data on nephron formation in the fetal human kidney during the late phase of gestation is a call to start with intense research so that concepts for a therapeutic prolongation of nephrogenesis can be designed.

Mathematical models for kidney function focusing on clinical interest.

We give an overview of mathematical models of renal physiology and anatomy with the clinician in mind. Beyond the past focus on issues of local transport mechanisms along the nephron and the urine concentrating mechanism, recent models have brought insight into difficult problems such as renal ischemia (oxygen and CO2 diffusion in the medulla) or calcium and potassium homeostasis. They have also provided revealing 3D reconstructions of the full trajectories of families of nephrons and collecting ducts through cortex and medulla. The recent appearance of sophisticated whole-kidney models representing nephrons and their associated renal vasculature promises more realistic simulation of renal pathologies and pharmacological treatments in the foreseeable future.

Estimation of nephron number in living humans by combining unenhanced computed tomography with biopsy-based stereology.

Methods for estimating nephron number in a clinical setting may be useful for predicting renal outcomes. This study aimed to establish such a method using unenhanced computed tomography (CT) and biopsy-based stereology. Patients or living kidney donors simultaneously subjected to enhanced and unenhanced CT examinations were randomly assigned to development and validation groups. The enhanced CT-measured arterial phase and the venous phase images of kidneys were regarded as the true values for cortical volume and parenchymal volume, respectively. Linear multiple regression analysis was used to create models for estimating cortical volume using explanatory variables including unenhanced CT-measured parenchymal volume. Nephron number was determined as the product of cortical volume and the glomerular density in biopsies of donors. Five equations for estimating cortical volume were created and verified. In donors, estimated nephron number by unenhanced CT was consistent with that by enhanced CT, with minimal errors in all models (636-655 ± 210-219 vs. 648 ± 224 × 103/kidney). Clinical characteristics combined with parenchymal volume did not improve the equation over parenchymal volume alone. These results support the feasibility of estimating nephron number by a combination of unenhanced CT and biopsy-based stereology, with a possible application for renal disease patients who are often not suitable for contrast media.

A large drawing of a nephron for teaching medical students renal physiology, histology, and pharmacology.

The purpose of this study is to see whether a large drawing of a nephron helped medical students in self-directed learning groups learn renal physiology, histology, and pharmacology before discussing clinical cases. The end points were the grades on the renal examination and a student survey. The classes in the fall of 2014 and 2015 used the drawing, but not those of 2012 and 2013. The Charles E. Schmidt College of Medicine at Florida Atlantic University is a newly formed Florida medical school, which enrolled its first class in the fall of 2011. The school relies on self-directed problem-based learning in year 1 and changes over to a case inquiry method in the latter part of year 1 and throughout year 2. At the start of the renal course, each student group received a poster of a nephron with the objective of learning the cell functions of the different nephron parts. During the first year of using the drawing, there was no improvement in grades. After a student suggested adjustment to the drawing, there was a statistically significant difference in the total test score in the second year ( P < 0.001). An unexpected finding was lower grades in all 4 yr in the area of acid-base balance and electrolytes compared with the other four areas tested. In the survey, the students found the drawing useful.

Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney.

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.

Reading First Coordinates from the Nephrogenic Zone in Human Fetal Kidney.

While substantial information is available on organ anlage and the primary formation of nephrons, molecular mechanisms acting during the late development of the human kidney have received an astonishing lack of attention. In healthy newborn babies, nephrogenesis takes place unnoticed until birth. Upon delivery, morphogenetic activity in the nephrogenic zone decreases, and the stem cell niches aligned beyond the organ capsule vanish by an unknown signal. However, this signal also plays a key role in preterm and low birth weight babies. Although they are born in a phase of active nephrogenesis, pathological findings illustrate that they evolve to a high incidence oligonephropathy and prematurity of renal parenchyma. Different extra- and intrauterine influences seem to be responsible, but independent from chemical nature, all of them culminate in the nephrogenic zone. One assumes that the marred development is caused either by an overshoot of metabolites, misleading signaling of morphogens, unbalanced synthesis of extracellular matrix or restricted contact between mesenchymal and epithelial stem cells. Even more surprising is that there is only a few vague morphological information of the nephrogenic zone in the human fetal kidney available and ultrastructural data is severely lacking. On this account, the first coordinates were determined by optical microscopy and morphometry. Without claiming to be complete, generated results made it possible to create schematic illustrations true to scale for orientation. It will help graduating students, young pediatricians, pathologists, and scientists working in the field of biomedicine to interpret professionally the nephrogenic zone and contained niches.

MITF - A controls branching morphogenesis and nephron endowment.

Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.

Estimated Nephron Number of the Donor Kidney: Impact on Allograft Kidney Outcomes.

BACKGROUND: Low birth weights have been associated with a reduction in nephron number with compensatory hypertrophy of existing glomeruli. The impact of donor birth weight as an estimate of nephron number on allograft function, however, has not been examined. METHODS: We collected donor birth weight, kidney weight, and volume from 91 living kidney donor-recipient pairs before nephrectomy and after 12, 36, and 60 months. Nephron number was calculated from donor birth weight and age. RESULTS: Donor birth weight, kidney weight/body surface area (BSA), and kidney volume showed a moderate positive correlation with allograft estimated glomerular filtration rate (eGFR) at 12 months (P < .05). Donor age showed a negative moderate correlation with allograft eGFR at 12 months (P = .015). The strongest correlation with allograft eGFR was observed for calculated donor kidney nephron number at 12, 36, and 60 months (R, 0.340, 0.305, and 0.476, respectively; P < .05). No impact was observed on allograft daily proteinuria of any investigated marker (P > .05). Recipients of donors with birth weight <2.5 kg had need of a significantly greater number of antihypertensive drugs (P < .05). CONCLUSIONS: Calculated nephron number from donor birth weight and age is suggested to be superior to donor kidney weight/BSA and volume regarding allograft function. Calculated nephron number could estimate expected eGFR and guide decision making in cases of impaired allograft function.

Preterm Birth and its Impact on Renal Health.

Preterm birth occurs in approximately 10% of all births worldwide. Preterm infants have reduced nephron numbers at birth in proportion to gestational age, and are at increased risk of neonatal acute kidney injury as well as higher blood pressure, proteinuria, and chronic kidney disease later in life. Rapid catch-up growth in preterm infants, especially if resulting in obesity, is a risk factor for end-stage kidney disease among children with proteinuric renal disease. Preterm birth, however, is a risk factor not only for the infant because mothers who deliver preterm have an increased risk of having subsequent preterm deliveries as well as hypertension, cardiovascular disease, and renal disease later in life. Preterm birth in a female infant is also a risk factor for her future risk of having a preterm delivery, gestational hypertension, and gestational diabetes, which in turn may impact the development of fetal kidneys and the offspring's risk of hypertension and renal disease. This intergenerational programming cycle, therefore, perpetuates the risks and consequences of prematurity. Interruption of this cycle may be possible through optimization of maternal nutrition and health as well as careful antenatal care, which may in turn reduce the global burden of hypertension and renal disease in subsequent generations.

Mapping the Nephron Exercise Incorporates Multiple Learning Strategies.

Introduction: Understanding the location and action of nephron transporters and channels is important to the understanding of renal function. As each region of the nephron is unique in its inclusion of specific transporters and channels, mapping of the nephron is an effective first step in understanding overall nephron processing. We describe a small-group, active-learning exercise that facilitates students' ability to understand renal processing within each region of the nephron. Methods: Following an overview lecture on renal transporters and channels, small groups of students worked cooperatively to map the nephron. This 2-hour, collaborative exercise was developed to reinforce key concepts in renal processing of ions and nutrients and, at the same time, utilize effective learning strategies. Learning strategies incorporated in this exercise include small-group collaboration, peer teaching, retrieval practice using an audience response system, and elaboration through discussion. Results: Written examination was used to assess student understanding. Students demonstrated higher performance on a subset of questions related to this learning activity compared to the overall exam. Highly positive feedback was provided by a convenience sample of students completing an anonymous survey. Discussion: This nephron-mapping exercise was an effective means to promote synthesis and analysis of lecture content and engage students in methods that enhance learning.

Caudal migration and proliferation of renal progenitors regulates early nephron segment size in zebrafish.

The nephron is the functional unit of the kidney and is divided into distinct proximal and distal segments. The factors determining nephron segment size are not fully understood. In zebrafish, the embryonic kidney has long been thought to differentiate in situ into two proximal tubule segments and two distal tubule segments (distal early; DE, and distal late; DL) with little involvement of cell movement. Here, we overturn this notion by performing lineage-labelling experiments that reveal extensive caudal movement of the proximal and DE segments and a concomitant compaction of the DL segment as it fuses with the cloaca. Laser-mediated severing of the tubule, such that the DE and DL are disconnected or that the DL and cloaca do not fuse, results in a reduction in tubule cell proliferation and significantly shortens the DE segment while the caudal movement of the DL is unaffected. These results suggest that the DL mechanically pulls the more proximal segments, thereby driving both their caudal extension and their proliferation. Together, these data provide new insights into early nephron morphogenesis and demonstrate the importance of cell movement and proliferation in determining initial nephron segment size.

Lengths of nephron tubule segments and collecting ducts in the CD-1 mouse kidney: an ontogeny study.

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.

Modeling of Kidney Hemodynamics: Probability-Based Topology of an Arterial Network.

Through regulation of the extracellular fluid volume, the kidneys provide important long-term regulation of blood pressure. At the level of the individual functional unit (the nephron), pressure and flow control involves two different mechanisms that both produce oscillations. The nephrons are arranged in a complex branching structure that delivers blood to each nephron and, at the same time, provides a basis for an interaction between adjacent nephrons. The functional consequences of this interaction are not understood, and at present it is not possible to address this question experimentally. We provide experimental data and a new modeling approach to clarify this problem. To resolve details of microvascular structure, we collected 3D data from more than 150 afferent arterioles in an optically cleared rat kidney. Using these results together with published micro-computed tomography (µCT) data we develop an algorithm for generating the renal arterial network. We then introduce a mathematical model describing blood flow dynamics and nephron to nephron interaction in the network. The model includes an implementation of electrical signal propagation along a vascular wall. Simulation results show that the renal arterial architecture plays an important role in maintaining adequate pressure levels and the self-sustained dynamics of nephrons.