Morphometry: assessing direct and indirect methods of measuring the diameters of tubular structures / Morfometría: evaluación de métodos directos e indirectos para medir los diámetros de las estructuras tubulares

SUMMARY: Knowledge of the diameter of a structure or particle is required for stereological calculations. However, there is no consensus on the methodology for its measurement. This study aims to assess the differences between direct and indirect methods of measuring diameter. It is hypothesised that kidneys were removed, fixed, processed, sectioned, and stained. The stained slides were imaged using a digital microscope. The images were processed using the ImageJ software. The diameters of the renal glomeruli and collecting tubules were measured using direct and indirect methods. The measured diameters were analysed using the SPSS software v20. The differences between the measurements were assessed using a Z-test and test of association, and P < 0.05 was considered significant. No significant differences were observed between the diameters of the glomeruli (P = 0.82) and proximal (P = 0.86) and distal (P = 0.55) convoluted tubules as measured via direct and indirect methods. There was a strong positive correlation between the diameters of glomeruli (P = 0.97) and proximal (P = 0.82) and distal (P = 0.93) convoluted tubules measured using the two methods, both of which are convenient, accurate and suitable. The P-values based on these measurements were more than 0.05. Therefore, the study hypothesis was rejected. There was no significant difference between the direct and indirect methods of measuring diameter, and the null hypothesis was rejected; thus, both methods can be applied either independently or jointly.

RESUMEN: Se requiere el conocimiento del diámetro de una estructura o partícula para los cálculos estereológicos. Sin embargo, no existe consenso sobre la metodología para su medición. Este estudio tuvo como objetivo evaluar las diferencias entre los métodos directos e indirectos de medición del diámetro de una estructura. Riñones de ratas Wistar fueron extirpados, fijados, procesados y seccionados, y luego se tiñeron con HE. Se tomaron imágenes de las muestras teñidas usando un microscopio digital. Las imágenes fueron procesadas utilizando el software ImageJ. Los diámetros de los glomérulos renales y túbulos colectores se midieron por métodos directos e indirectos. Los diámetros medidos se analizaron utilizando el software SPSS v20. Las diferencias entre las medidas se evaluaron mediante una prueba Z y una prueba de asociación, y se consideró significativa P < 0,05. No se observaron diferencias significativas entre los diámetros de los glomérulos (P = 0,82) y túbulos contorneados proximales (P = 0,86) y distales (P = 0,55) medidos mediante métodos directos e indirectos. Hubo una fuerte correlación positiva entre los diámetros de los glomérulos (P = 0,97) y los túbulos contorneados proximales (P = 0,82) y distales (P = 0,93) medidos con los dos métodos, ambos convenientes, precisos y adecuados. Los valores P basados en estas mediciones fueron superiores a 0,05. Por lo tanto, se rechazó la hipótesis del estudio. No hubo diferencia significativa entre los métodos directo e indirecto de medición del diámetro, y se rechazó la hipótesis nula; por lo tanto, ambos métodos se pueden aplicar de forma independiente o conjunta.

Brief Synopsis: Review of Renal Tubule and Interstitial Anatomy and Physiology and Renal INHAND, SEND, and DIKI Nomenclature.

This article provides a synopsis of the first two presentations from the second scientific session of the 37th Annual Symposium of the Society of Toxicologic Pathology in Indianapolis, Indiana, on June 18, 2018; the session focused on acute kidney injury. The first presentation, given by Dr. Kevin McDorman, focused on "Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology." Several common background findings from toxicity studies were additionally discussed. Lastly, factors that impact the relevance and usefulness of historical control data, such as quality and consistency of histopathology, were discussed. The second presentation, given by Dr. Torrie Crabbs, provided a review of International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), Standard for Exchange of Nonclinical Data (SEND), and drug-induced kidney injury (DIKI) nomenclature. INHAND is a global collaborative project that provides internationally accepted standardized nomenclature and diagnostic criteria for proliferative and nonproliferative changes in laboratory animals in toxicity and carcinogenicity studies. SEND is currently a required standard for data submission to the Food and Drug Administration (FDA). Since the FDA has indicated its preference for INHAND nomenclature, SEND will predominately use INHAND terminology; thus, familiarity with INHAND terminology is critical for toxicologic pathologists. The diagnostic features of three common DIKI findings, in addition to several complicated INHAND terminologies, were reviewed.

Molecular phenotypes of the human kidney: Myoid stromal cells/telocytes and myoepithelial cells.

The connective stromal and epithelial compartments of the kidney have regenerative potential and phenotypic flexibility. A few studies have shown that cells appertaining to both compartments can exhibit myoid phenotypes. The purpose of our study was to investigate the myoid pattern of kidney and its association with the kidney niches containing stromal cells/telocytes (SC/TCs). We performed an immunohistochemical study using a panel of endothelial, myoid, mesenchymal and stem/progenitor markers, namely CD31, CD34, CD105 (endoglin), CD117/c-kit, nestin, desmin, α-smooth muscle actin (α-SMA) and the heavy chain of smooth muscle myosin (SMM). We used histologically normal kidney samples, obtained after nephrectomy, from nine adult patients. The capsular SC/TCs had a strong CD34 and partial nestin and CD105 immunopositivity. Subcapsular and interstitial SC/TCs expressed c-kit, nestin, CD105, but also α-SMA and SMM, therefore having a myoid phenotype. The endothelial SC/TCs phenotype was CD31+/CD34+/CD105+/nestin±/SMM±/α-SMA±. All three myoid markers were expressed in periendothelial SC/TCs. We also found a scarce expression of nestin in parietal epithelial cells of Bowman's capsule, and in podocytes. In epithelial cells, we found a positive expression for CD31, CD117/c-kit, desmin, CD34, SMM, and CD105. In epithelial tubular cells, we found a predominant basal expression of the myoid markers (SMM and desmin). In conclusion, myoepithelial tubular cells, myoid endothelial cells and myoid SC/TCs are normal constituents of the kidney.

Rho GAP myosin IXa is a regulator of kidney tubule function.

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.

Assessment of glomerular and tubular function.

At birth, GFR and tubular function of neonates is compromised as compared to older children and adults. These functions are even less developed in premature infants. These facts have a direct bearing on drug dosing, fluid and electrolyte administration, and maintenance of acid-base balance in neonates. Although many detailed methods of assessing renal functions have been provided in this article, laboratory and radiologic studies available in most healthcare facilities are often sufficient to provide a clinically relevant data in most patients, including neonates.

Tubular control of renin synthesis and secretion.

The intratubular composition of fluid at the tubulovascular contact site of the juxtaglomerular apparatus serves as regulatory input for secretion and synthesis of renin. Experimental evidence, mostly from in vitro perfused preparations, indicates an inverse relation between luminal NaCl concentration and renin secretion. The cellular transduction mechanism is initiated by concentration-dependent NaCl uptake through the Na-K-2Cl cotransporter (NKCC2) with activation of NKCC2 causing inhibition and deactivation of NKCC2 causing stimulation of renin release. Changes in NKCC2 activity are coupled to alterations in the generation of paracrine factors that interact with granular cells. Among these factors, generation of PGE2 in a COX-2-dependent fashion appears to play a dominant role in the stimulatory arm of tubular control of renin release. [NaCl] is a determinant of local PG release over an appropriate concentration range, and blockade of COX-2 activity interferes with the NaCl dependency of renin secretion. The complex array of local paracrine controls also includes nNOS-mediated synthesis of nitric oxide, with NO playing the role of a modifier of the intracellular signaling pathway. A role of adenosine may be particularly important when [NaCl] is increased, and at least some of the available evidence is consistent with an important suppressive effect of adenosine at higher salt concentrations.

Mechanical instabilities of biological tubes.

We study theoretically the morphologies of biological tubes affected by various pathologies. When epithelial cells grow, the negative tension produced by their division provokes a buckling instability. Several shapes are investigated: varicose, dilated, sinuous, or sausagelike. They are all found in pathologies of tracheal, renal tubes, or arteries. The final shape depends crucially on the mechanical parameters of the tissues: Young's modulus, wall-to-lumen ratio, homeostatic pressure. We argue that since tissues must be in quasistatic mechanical equilibrium, abnormal shapes convey information as to what causes the pathology. We calculate a phase diagram of tubular instabilities which could be a helpful guide for investigating the underlying genetic regulation.

[Renal physiology]. / Physiologie rénale.

The kidneys are responsible for the urinary excretion of uremic toxins and the regulation of several body systems such as intra and extracellular volume status, acid-base status, calcium and phosphate metabolism or erythropoiesis. They adapt quantitative and qualitative composition of the urine to keep these systems in balance. The flow of plasma is filtered in the range of 120 mL/min, and depends on the systemic and renal hemodynamics which is subject to self-regulation. The original urine will then be modified in successive segments of the nephron. The proximal nephron is to lead the massive reabsorption of water and essential elements such as sodium, bicarbonates, amino-acids and glucose. The distal nephron includes the distal convoluted tubule, the connector tube and the collecting duct. Its role is to adapt the quality composition of urine to the needs of the body.

Endotoxin uptake by S1 proximal tubular segment causes oxidative stress in the downstream S2 segment.

Gram-negative sepsis carries high morbidity and mortality, especially when complicated by acute kidney injury (AKI). The mechanisms of AKI in sepsis remain poorly understood. Here we used intravital two-photon fluorescence microscopy to investigate the possibility of direct interactions between filtered endotoxin and tubular cells as a possible mechanism of AKI in sepsis. Using wild-type (WT), TLR4-knockout, and bone marrow chimeric mice, we found that endotoxin is readily filtered and internalized by S1 proximal tubules through local TLR4 receptors and through fluid-phase endocytosis. Only receptor-mediated interactions between endotoxin and S1 caused oxidative stress in neighboring S2 tubules. Despite significant endotoxin uptake, S1 segments showed no oxidative stress, possibly as a result of the upregulation of cytoprotective heme oxygenase-1 and sirtuin-1 (SIRT1). Conversely, S2 segments did not upregulate SIRT1 and exhibited severe structural and functional peroxisomal damage. Taken together, these data suggest that the S1 segment acts as a sensor of filtered endotoxin, which it takes up. Although this may limit the amount of endotoxin in the systemic circulation and the kidney, it results in severe secondary damage to the neighboring S2 segments.

Kidney development: two tales of tubulogenesis.

The mammalian kidney may well be one of the most complex organs of postnatal life. Each adult human kidney contains on average more than one million functional filtration units, the nephrons, residing within a specialized cellular interstitium. Each kidney also contains over 25 distinct cell types, each of which must be specifically aligned with respect to each other to ensure both normal development and ultimately, normal renal function. Despite this complexity, the development of the kidney can be simplistically described as the coordinate formation of two distinct sets of tubules. These tubules develop cooperatively with each other in time and space, yet represent two distinct but classical types of tubulogenesis. The first of these tubules, the ureteric bud, forms as an outgrowth of another epithelial tube, the nephric duct, and undergoes extensive branching morphogenesis to create the collecting system of the kidney. The second tubules are the nephrons themselves which arise via a mesenchyme-to-epithelial transition induced by the first set of tubules. These tubules never branch, but must elongate to become intricately patterned and functionally segmented tubules. The molecular drivers for these two tales of tubulogenesis include many gene families regulating tubulogenesis and branching morphogenesis in other organs; however, the individual players and codependent interrelationships between a branched and non-branched tubular network make organogenesis in the kidney unique. Here we review both what is known and remains to be understood in kidney tubulogenesis.

Alteraciones en las características morfométricas del riñón de ratas albinas machos provocadas por la ingestión crónica de etanol desde la adolescencia / Alterations in the morphometric features of kidney of male albino rats provoked by chronic ingestion of ethanol from the adolescence

En el presente trabajo se pretendió determinar las variaciones que sobre las características morfométricas del riñón provoca la ingestión crónica de etanol en ratas adolescentes, para lo cual se utilizaron 42 ratas albinas machos de 30 días de nacidas con las cuales se conformaron dos grupos de 21 animales cada uno y con tiempos de tratamiento de tres y cinco meses y con estos, dos subgrupos: experimental y control. A las ratas experimentales se les suministró etanol a dosis de 5 g/kg de peso corporal mediante cánula intraesofágica. A las controles se les administró agua en lugar de etanol, en iguales condiciones. Se emplearon cortes histológicos coloreados con técnica PAS y se estudiaron las porciones contorneadas de los túbulos proximales y distales. Se calculó el área de sección transversal tubular y se midieron los volúmenes nucleares de las células de ambos túbulos. Se comprobó que las ratas experimentales mostraron volúmenes nucleares menores que las controles. El grupo experimental mostró valores de áreas de sección transversal de los túbulos renales mayores que los controles, siendo estos valores superiores en los túbulos proximales en las ratas de cinco meses, y los distales en las ratas de tres meses. Se concluyó que en la muestra estudiada el alcoholismo crónico iniciado en la adolescencia provocó variaciones morfométricas en los túbulos proximales y distales del riñón

Present paper allowed us to determine the variations exerted by the chronic ingestion of ethanol from the adolescence on the kidney morphometric features in 42 male albino rats of 30 days born divided into two groups of 21 animal each and with the treatment times of three and five months and with these 42 animals into two subgroups: one of experimental type and other as control. The experimental rats received ethanol at 5 g/kg doses according to the body weight using an intraesophageal cannula. The control ones received water instead of ethanol in similar conditions. We used histological sections stained with PAS technique to study the outlined portions of the proximal and distal tubules. The tubular transversal section area was estimated measuring the nuclear volumes of cells in both tubules. It was demonstrated that the experimental rats showed higher values of the transversal section of renal tubules higher than the control ones, where these values were superior in proximal tubules in the five months old rats and the distal ones in the three months old. We conclude that in study sample the chronic alcoholism started during adolescence provoked morphometric variations in proximal and distal tubules of kidney

Tangential Algorithm for Calculation of the Fractal Dimension of Kidney Tubuli Sections / Algoritmo de Tangencialización para el Cálculo de la Dimensión Fractal de Túbulos Renales Seccionados

We have developed a new algorithm to calculate the fractal dimension of dog kidney proximal convoluted tubule section. This algorithm is intermediate between box-counting and perimeter-stepping algorithms. The result of this new algorithm and correlations found between fractal dimensions and other Euclidian values of these tubuli, suggest that this algorithm is adequate to calculate fractal dimensions by points.

Hemos desarrollado un nuevo algoritmo para calcular la dimensión fractal de las secciones de los túbulos contorneados proximales del riñón de perro. Este algoritmo es promedio de los que emplean el recuento de cajas y la medida del perímetro. El resultado de las medidas con este nuevo algoritmo y las correlaciones encontradas entre sus valores de dimensión fractal y otros valores euclidianos de dichos túbulos, sugieren la adecuación de método para calcular la dimensión fractal referida a puntos.

Renal tubular damage detected by dynamic micro-MRI with a dendrimer-based magnetic resonance contrast agent.

BACKGROUND: A noninvasive technique to evaluate the structure and function of the kidney would be useful to investigate renal diseases, especially acute renal failure. We have developed a novel technique to visualize functional micro-magnetic resonance (MR) images of the mouse kidney with a dendrimer-based macromolecular renal MR contrast agent. METHOD: Mice were injected with cisplatin or vehicle, then examined three days later by contrast-enhanced, dynamic high-resolution micro-MRI with 160 microm spatial resolution using a 1.5 T clinical MRI unit, a surface coil, and the renal contrast agent G4D-(1B4M-Gd)64. RESULTS: The cortex and outer stripe of the outer medulla of the mouse kidney were clearly visualized in the normal mice. In animals treated with cisplatin, the gradation of tubular damage as assessed by contrast enhanced dynamic MRI correlated with renal function. CONCLUSION: Contrast-enhanced, dynamic high-resolution micro-MRI with a novel dendrimer-based macromolecular renal MR contrast agent can be a powerful tool for in vivo observation of renal structural and functional damage.

Pathological expression of renin and angiotensin II in the renal tubule after subtotal nephrectomy. Implications for the pathogenesis of tubulointerstitial fibrosis.

The finding that the systemic renin-angiotensin system (RAS) is not activated in most types of chronic renal disease has led to the suggestion that a local, intrarenal RAS may be an important determinant in the relentless progression of renal disease. Therefore, cell specific changes in various components of the RAS in response to renal mass reduction and angiotensin converting enzyme (ACE) inhibition were examined. Thirty Sprague-Dawley rats were randomly assigned to sham surgery, subtotal nephrectomy (STNx) alone or STNx treated with the ACE inhibitor, perindopril, and sacrificed after 12 weeks. In sham rats, renin mRNA and protein were only present in the juxtaglomerular apparatus. In contrast, in STNx kidneys, renin and angiotensin II expression were noted predominantly in renal tubular epithelial cells in association with overexpression of the prosclerotic cytokine, transforming growth factor-beta1 (TGF-beta1). In perindopril-treated STNx rats expression of renin and TGF-beta1 were similar to control animals. These finding indicate that following renal mass reduction there is pathological tubular expression of various components of the RAS. Furthermore, in contrast to the juxtaglomerular apparatus, tubular renin expression was reduced with ACE inhibition. These changes within the intrarenal RAS may be pathogenetically linked to the development of tubulointerstitial injury.

Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4.

The kidney has been widely exploited as a model system for the study of tissue inductions regulating vertebrate organogenesis. Kidney development is initiated by the ingrowth of the Wolfian duct-derived ureteric bud into the presumptive kidney mesenchyme. In response to a signal from the ureter, mesenchymal cells condense, aggregate into pretubular clusters and undergo an epithelial conversion generating a simple tubule. This then undergoes morphogenesis and is transformed into the excretory system of the kidney, the nephron. We report here that the expression of Wnt-4, which encodes a secreted glycoprotein, correlates with, and is required for, kidney tubulogenesis. Mice lacking Wnt-4 activity fail to form pretubular cell aggregates; however, other aspects of mesenchymal and ureteric development are unaffected. Thus, Wnt-4 appears to act as an autoinducer of the mesenchyme to epithelial transition that underlies nephron development.

Differences in rat kidney morphology between males, females and testosterone-treated females.

Kidneys of normal female and male Wistar-Kyoto rats were studied by standard morphological techniques and morphometry in order to evaluate possible differences in the overall kidney morphology between both sexes. Furthermore, we investigated the role of testosterone (DHT) on kidney morphology by treating females with daily DHT injections. Kidney weight and volume in relation to body weight were not significantly different between males and females and were not affected by DHT. Differences were found in the volume distribution among the kidney zones. The cortex was larger in males than in females, whereas the medulla was conspicuously larger in females than in males. The greater volume of the cortex in males was mainly due to a more extensive development of proximal tubules. DHT treatment in females increased the volume of their proximal tubules. Glomerular volume was similar among the three groups. Within the medulla, the difference was most prominent in the inner stripe (14.9% of the total kidney volume in females vs. 8.9% in males) and was also important in the inner medulla (7.0 vs. 4.8%). The absolute epithelial volume of thick ascending limbs in this zone was larger in females than in males. This difference was more pronounced in short loops (approximately 20%) than in long loops (approximately 10%). The values of the DHT-treated females ranged in between. In spite of the greater development of medulla and thick ascending limbs in females, urine concentration was higher in males than in females and maximum urinary concentrating ability after 48 h dehydration was not different between both sexes.

Variable NMR visibility of intracellular sodium induced by Na(+)-substrate cotransport in dog cortical tubules.

The intracellular sodium concentration [( Na+]i) of dog kidney cortical tubules was monitored by flame photometry and 23Na NMR using dysprosium tripolyphosphate as shift reagent. Upon addition of substrates cotransported with sodium, flame photometry showed an increase in [Na+]i while no change (glutamine, glucose) or even a decrease (lactate) in the Na+i NMR signal was observed. This discrepancy could not be explained by a lack of ATP prior to the addition of substrates or by a decrease of NMR visibility of Nai+ induced by binding of substrate to membrane transporters (and pump). We propose that a variation of the "apparent visibility" of Nai+ may occur, arising from either a compartmentation of Nai+ in dog cortical tubules or an inhomogeneous extracellular distribution of the shift reagent.

Diluting segment in kidney of dogfish shark. I. Localization and characterization of chloride absorption.

Single tubules, dissected from the peritubular sheath of the dorsal bundle zone of kidney of the dogfish shark, Squalus acanthias, were perfused in vitro at 17-18 degrees C. This segment is the largest of the five in the peritubular sheath and had average inner and outer diameters of 46.9 +/- 1.2 and 74.4 +/- 2.1 microns, respectively (n = 32). These values suggest that this is the intermediate IV segment. When perfused with symmetrical buffered elasmobranch saline, intermediate IV segments exhibited high rates of Cl- absorption (JCl, pmol.s-1.cm-2): 1,696 at an average perfusion rate (Vo) of 8.2 nl/min. Cl- absorption was highly flow dependent [1/JCl = 57.95(1/Vo) + 1.75; r = 0.71, P less than 0.01]. Maximal rates of Cl-absorption, calculated from reciprocal transformation of the flow dependence of JCl, yielded a value of 5,714 pmol.s-1.cm-2. In the presence of a 200-mosmol/kg transepithelial osmotic gradient, fluid absorption was negligible. The spontaneous transepithelial voltage (Vte, mucosal with respect to serosal compartment) averaged 8.0 +/- 1.0 mV (n = 26). Such active transport of Cl- in the absence of fluid movement and in the presence of a lumen-positive transepithelial voltage is characteristic of amphibian and mammalian diluting segments. Na(+)-to-Cl- permeability ratios (PNa/PCl) averaged 2.5 +/- 0.5, indicating that, as in mammalian thick ascending limbs, this segment is Na+ (cation) permselective. Vte was dependent on the presence of Na+ and Cl- in the external solutions and was reversibly abolished by isosmotic replacement with N-methyl-D-glucamine or with isethionate, respectively. Ouabain inhibited Vte but was not reversible within the time course of these experiments. Furosemide (10(-4) M), but not equimolar concentrations of amiloride or hydrochlorothiazide, added to the luminal perfusate inhibited both Vte and JCl. These results suggest that apical membrane Na+ entry in intermediate IV segments is mediated by Na(+)-K(+)-Cl- cotransport and is consistent with the existence of a functional role of urinary dilution in the reabsorption of urea in the elasmobranch kidney.