Fluorescent Labeling Approach for Visualizing and Quantifying Glomerular Density and Volume in Mouse Kidneys

Background and Aims The aim of the study was to evaluate glomerular morphometry in obese and non-obese patients with glomerular diseases: FSGS, IgA nephropathy, membranous glomerulonephritis (MGN) and membranoproliferative glomerulonephritis (MPGN). Method The study included 125 patients with glomerular diseases (77 males, mean age 46± 15.2 years): obese (BMI> 27kg/m2- 63 patients) and non-obese (BMI< 27kg/m2-62 patients). The serum concentration of creatinine, proteins, albumin, cholesterol, trygliceride, daily proteinuria were measured at the time of kidney biopsy. Formula Cockcroft-Gault#(Cockcroft-Gault: non-obese) and Cockcroft-GaultLBW: obese) was calculated. Glomerular radius (GR), glomerular volume (GV) and glomerular density (GD) were compared morphometrically between two groups. Results At the time of kidney biopsy the obese had significantly lower eGFR, significantly higher creatinine, trygliceride, daily proteinuria compared to non-obese. Obese FSGS patients had significantly higher GR (109.44±6.03 μm vs 98.53±14.38 μm) and GV (3.13±0.49 x106 μm?vs 2.26±0.83 x106 μm?, only midly lower GD (1.91±0.39/mm2 vs 1.95±0.61/mm2) compared to non-obese. Obese IgA nephropathy patients had higher GR (110.69± 15.24 μm vs 107.77±14.53 μm), significantly higher GV(3.34±1.78 x106 μm?vs 2.31± 1.51 x106 μm? and significantly lower GD (1.21±0.29/mm2 vs 2.14±0.54/mm2) in comparation with non-obese. Significant positive association between GV and BMI (r=0.439; p<0.01) in FSGS patients was found. Significant positive correlation between GV and BMI (r=0.683; p< 0.01), but significant negative correlation between GD and BMI (r=-0.710; p< 0.01) in IgA nephropathy patients were found. Obese MGN patients had lower GR (112.82± 13.65 μm vs 118.39± 14.52 μm), GV (3.57± 1.19 x106 μm?vs 4.07± 1.28 x106 μm? and GD (1.89± 0.17/ mm2 vs 1.91± 0.92/mm2) but without significance compared to non-obese. Obese MPGN patients had higher GR(105.89± 12.24 μm vs 92.73± 30.01 μm), GV (2.94± 0.91 x106 μm?vs 2.31± 1.27 x106 μm? and GD (2.32± 0.63/ mm2 vs 2.03± 0.73/ mm2) but without significance in comparation with non-obese. Significant negative association between GD and daily proteinuria (r=-0.417; p<0.01) in MGN patients was found. Conclusion Obese FSGS patients had significantly higher GV, lower GD compared to non-obese. Obese IgA nephropathy patients had significantly higher GV and significantly lower GD. Obese MGN patients had lower and obese MPGN patients had higher morphometric parameters of glomeruli compared to non-obese. Significant positive correlation between GV and BMI in FSGS and IgA nephropathy patients were found.

The role of the kidney in the pathogenesis of hypertension has long been established, although recent studies challenge renal hegemony and suggest an important role for vascular cells as well (1,2). In recent years, the formulation has expanded and now includes the concept that chronic hypertension and kidney disease are also related to events that occur during the prenatal period and usually result in low birth weight (LBW). A recent overview considered the role of fetal programming in the development of adult kidney disease and hypertension (3). The aim of this review is to present further evidence to support an association between LBW and the increased prevalence of hypertension as well as progression toward chronic kidney disease (CKD) in adult life. Various potential mechanisms for this association are discussed, specifically the low nephron number (LNN) hypothesis and related cellular and molecular mechanisms that have been proposed. LBW infants are defined as infants who weigh ≤2500 g at birth. Infants who are delivered before 37 wk from the first day of the last menstrual period are termed preterm. The LBW infant population can be divided into preterm, appropriate for gestational age (AGA), or small for gestational age (SGA). The predominant cause of LBW infants in the United States is preterm birth, whereas in developing countries, the cause is often intrauterine growth restriction (IUGR). During the year 2004, 8.1% of births in the United States were LBW, and more than half of these were preterm. During the past two decades, there has been an increase in the prevalence of LBW as higher risk pregnancies progress to term and postnatal survival improves (4) (Figure 1). Figure 1. Percentage of infants who were born preterm and percentage who were born with low birth weight (LBW; defined in text): United States, 1990, 1995, 2000, …

Background and Aims Recent studies have conflicting findings on the association between obesity and the risk of chronic kidney disease (CKD). BMI by itself is an imperfect marker of metabolically unhealthy obesity. The Pulse Mass Index (PMI) and the Pulse Mass Pressure Product (PMPP) show strong correlation with the risk of cardiovascular disease and may reflect an individual's metabolic energy state. However, it is still unknown whether the PMI and PMPP can be helpful to evaluate glomerular hyperfiltration or hypertrophy, which may exacerbate the progression of CKD. In this study, we measured mean glomerular volume (GV) and single nephron glomerular filtration rate (SNGFR) by estimating the number of nephrons from living kidney donors and we evaluated the effects of PMI and PMPP on glomerular hemodynamic function. Method We identified 43 living kidney donors at the Jikei University School of Medicine Hospital from 2007 to 2017 who underwent an enhanced CT scan and a kidney biopsy at donation. The GV was determined by Weibel-Gomez methods and the SNGFR was calculated as eGFR divided by the total number of non-sclerosed glomeruli. The PMI is equal to BMI by resting heart rate (RHR) divided by 1730 and The PMPP is equal to BMI by RHR by systolic blood pressure. Results Of 43 subjects, 34.9% were males and with a mean age of 56.1 ± 9.9 years old. Mean Arterial Pressure (MAP) was 86.3±9.9 mmHg, eGFR was 77.4±12.4 ml/min/1.73m2, and urinalysis was normal. The estimated total nephron numbers were 743,204±242,020 /kidney, and SNGFR was 61.7±20.5 nl/min/1.73m2. The GV was strongly associated with both of PMI and PMPP (P=0.0291 and 0.0326, respectively). Interestingly, SNGFR was only associated with PMPP (P=0.0443) but whereas not associated with either age, BMI, or MAP. Conclusion In healthy adults, we observed a strong positive correlation between PMPP and both GV and SNGFR. This study indicates that PMPP is a more suitable physical measurement for glomerular hyperfiltration or hypertrophy, and a possible maker of the relative risk of CKD at the long term.

We have previously shown that certain aspects of internephron heterogeneity are reduced or absent in Brattleboro rats with hereditary diabetes insipidus (DI) lacking ADH and can be restored by long-term ADH administration started before complete kidney maturation. In the present study, the effects of long- and short-term availability of ADH in adulthood were studied in Brattleboro DI rats. Single nephron glomerular filtration rate (SNGFR), glomerular volume (GV), and proximal tubular length (PTL) were measured in superficial and juxtamedullary nephrons using the ferrocyanide and microdissection techniques. ADH administration for 6 wk in adult DI rats (group A) restored normal nephron heterogeneity of SNGFR, GV, and PTL by increasing the filtration and size of deep nephrons. Acute changes in ADH availability induced either by 1-h ADH infusion in DI rats (group C) or by ADH discontinuation for 2 days in treated DI rats (group D) did not significantly change the anatomical parameters and only moderately affected SNGFR compared with the preexisting states (groups B and A, respectively). These results suggest that the influence of ADH on internephron heterogeneity is initiated by an increase in deep nephron SNGFR. Based on recent findings concerning the effects of ADH on the medullary (M) part of the thick ascending limbs (TAL), we suggest that the increase in deep nephron SNGFR after ADH may be due to a change in the tubuloglomerular feedback signal at the macula densa resulting from ADH-induced stimulation of the solute reabsorption in the MTAL. Superficial nephrons would be less sensitive to this change due to their long cortical TAL, which removes the macula densa further from the MTAL and provides additional sites for solute reabsorption.

A basis for accelerated progression of diabetic nephropathy in Pima Indians

The glomerular volumes in the kidneys were studied in four species of mammals, including a desert rodent, the jerboa ( Jaculus jaculus ); the laboratory white rat ( Rattus norvegicus ); a semidesert-living subprimate, the "bush baby" ( Galago senegalensis senegalensis ); and a primate, the grivet monkey ( Cercopitheus aethiops aethiops ). The measurements of volume were made according to the method of Palkovits and Zolnai which gives quantitative statistical values for the relative volumes of the glomeruli. An exact relationship between the average of the glomerular volumes, kidney weight, and body weight was not found in these different mammals when compared to each other. If the average volumes of the cortical and of the juxtamedullary glomeruli are compared separately then it is seen that the desert-living animals have a much bigger difference in volume between the cortical and the juxtamedullary glomeruli than those animals which require free access to water. In the kidney of the desert rodent (jerboa) the difference in volume between the cortical and the juxtamedullary glomeruli is 101% and, in the semidesert-living "bush baby," 169%. On the other hand the corresponding difference of volume in the laboratory white rat is only 21% and in the grivet monkey 28%. Considering the great functional importance of the juxtamedullary glomeruli and deep nephrons of the kidney in relation to dehydration, the large size of these structures can be regarded as an adaptation to life in an arid environment.

The development of single nephron glomerular filtration rate (SNGFER) was studied in both superficial and juxtamedullary nephrons in rats in relation to concomitant morphologic maturation. These experiments were carried out in rats between 23 and 91 days of age (between 36 nad 275 g body weight) with the [14C]ferrocyanide infusion technique. 2. SNGFR of the superficial and juxtamedullary nephrons increased with body weight, glomerular volume and proximal tubular length. 3. The ratio SNGFR of the superficial (S) nephrons/SNGFR of the juxtamedullary (JM) nephrons rose from 0-60 in the 40-60 g rats to 0-84 in the adult rats, demonstrating the centrifugal functional maturation of the nephrons. 4. The S/JM ratio for both glomerular volume and tubular length was constant and averaged 0-72+/--0-12 and 0-81+/-0-05, respectively, indicating that while the increase in SNGFR was greater for S than for JM nephrons, this was not accompanied by concomitant disproportionate increases of glomerular volume and/or proximal tubular length between these nephron categories during development in the rat.

BackgroundAmong the proteinuric patients with chronic kidney disease (CKD) who undergo a renal biopsy, we sometimes encounter those who cannot be classified as having a known primary or secondary glomerular disease. The pathogenesis and pathophysiology of these CKD patients have not been sufficiently elucidated.MethodsWe recruited 34 proteinuric patients without known glomerular diseases. The glomerular volumes (GV) of the biopsy specimens from those patients were determined by a morphometric analysis. Glomerular hypertrophy (GH) was defined as having more than 3.6 × 106 μm3. The patients were divided in two groups: those with GH (Group 1) and those without GH (Group 2). We compared the clinical and pathological parameters between Group 1 and Group 2, and among the three groups of patients: non-obese, overweight and obese group.ResultsThe patients with Group 1 had significantly higher values for the proportion of males, the body mass index (BMI), uric acid and significantly lower values for the glomerular density (GD). Of note, a multivariate regression analysis revealed that sex, the BMI and GD were significant factors correlated with the mean GV. The values for the mean GV were significantly higher in the overweight and obese groups as compared to the non-obese group, and the values for the GD were significantly lower in the obese group than in the non-obese group.ConclusionsWe identified a subgroup of patients who were characterized as having a high BMI and GV, and a low GD among the proteinuric CKD patients without known glomerular diseases.

reply: In aging male Wistar rats, renal injury develops spontaneously ([2][1]). It is gratifying that Dr. Schreuder is intrigued by our recent finding of a protective effect of early postnatal protein restriction (i.e., during lactation) on the spontaneous development of renal injury in male Wistar

Effects of growth hormone and IGF-I on glomerular ultrafiltration in growth hormone-deficient rats.

Our studies have demonstrated that a low glomerular density in renal biopsies is a plausible predictor of a worse renal outcome in patients with primary glomerular diseases. However, there remains a concern regarding the diversity that may exist in the distribution of glomerular density within the same kidney. This study therefore aimed to determine the differences in the glomerular density between anatomically different cortical zones of the human kidney. A total of 89 autopsy kidneys were analyzed to accurately measure the glomerular density in different parts of the renal cortex. As a whole, compared to the glomerular density in the superficial cortex (3.0 ± 0.7/mm(2)), the average glomerular density in the juxtamedullary cortex (2.2 ± 0.6/mm(2)) was approximately two-thirds. The glomerular density showed maximal 3.5-fold variations between individuals and was inversely correlated with the mean glomerular volume in both cortical areas. A low glomerular density of the superficial cortex was predominantly associated with the increase of global glomerulosclerosis. On the other hand, a low glomerular density of the juxtamedullary cortex was predominantly associated with an increase in the kidney weight. Thus, there are significant zonal differences in the distribution of the glomerular density in human kidneys independent of the potential variations observed between individuals.

Diabetes and hypertension share renal histopathological features, such as arterial lesions and glomerular hypertrophy, that have not been investigated in relation to the blood pressure status of diabetic subjects. The severity of glomerular lesions varies across locations of the renal cortex, which may be further affected by diabetes and/or hypertension. Histopathological lesions in different parts of the renal cortex of autopsy kidneys were evaluated and analyzed based on medical histories of diabetes and hypertension. This study included a total of 82 Japanese autopsies composed of normotensive nondiabetics (n = 31), hypertensive nondiabetics (n = 28), normotensive diabetics (n = 14), and hypertensive diabetics (n = 9). There were no differences in age, sex, renal function, or body size among groups. In both the superficial and juxtamedullary cortices, increased glomerular volume (GV) was significantly associated with either diabetes or hypertension. In addition, diabetes and hypertension showed a significant interaction with GV regardless of the cortical location. Values for global glomerulosclerosis (GGS) and arteriolar hyalinosis (AH) were associated with diabetes but not with hypertension. Only values for GGS consistently showed cortical surface superiority. The zonal distribution of AH, GV, or other diabetic glomerular lesions differed among the lesions depending on the presence or absence of hypertension. These results imply that diabetes and hypertension synergistically enhance glomerular hypertrophy across all layers of the human renal cortex. The process is closely associated with the severity of GGS and AH predominantly associated with diabetes.

To investigate the number of nephrons, using unbiased stereological method, after warm ischemia for laparoscopic partial nephrectomy in a pig model. Fourteen pigs underwent left laparoscopic partial nephrectomy, and four animals were used as controls (not operated). Renal vessels were clamped, and 25% of kidney length was resected. The animals were euthanized after 2 weeks, and the kidneys were collected. Thus, we analyzed 14 left kidneys submitted to warm ischemia, 14 right kidneys from the same animals but not submitted to ischemia and eight kidneys from control animals. Renal fragments were processed using routine histological methods. The total operative time and the time of warm ischemia were recorded. Glomerular mean volume and glomerular density were quantified by stereological methods. Creatinine serum levels were assessed preoperatively and before euthanasia. Surgical time was 71 ± 17 minutes, and ischemia time was 16 ± 5 minutes. The mean glomerular volume in the left kidneys was higher when compared with controls and to right kidneys (p<0.05). In addition, the glomerular density was reduced in the left kidneys (p<0.05) when compared with controls and right kidneys. Nevertheless, creatinine serum levels after 2 weeks of surgery were not different from the preoperative levels. No difference was found for stereological measurements between controls and right kidneys. Stereological determination of glomerular density can be used as an accurate and objective method for studies regarding renal damage from ischemia. Warm ischemia during laparoscopic partial nephrectomy in pigs determined a significant reduction of glomerular density in the ipsilateral remaining parenchyma.

The single nephron glomerular filtration rate (SNGFR) was determined in superficial (S) and juxtamedullary (JM) nephrons of 10 anesthetized rabbits by the 14C ferrocyanide infusion technique. The length of the proximal tubules and the volume of the glomeruli were also determined for the same nephrons. SNGFR was higher in JM than in S: 28.6 +/- 3.4 versus 22.6 +/- 3.0 nl/min, P less than 0.001. In JM nephrons, glomeruli were larger than in S: 1.3 +/- 0.1 versus 0.9 +/- 0.1 nl, P less than 0.001, whereas there was no difference between proximal tubule length in either category (s, 8.7 +/- 0.3 and JM, 8.9 +/- 0.5 mm). In 6 out of 8 animals, SNGFR was significantly correlated to glomerular volume. Lack of correlation was observed between SNGFR and length of proximal tubule in all animals but one. These results show that the rabbit, as well as small rodents and the dog, has a higher SNGFR in juxtamedullary than superficial glomeruli. Although this functional difference is not related to the length of the proximal tubule in each individual animal, the ratio between the mean SNGFR value and the mean length of the proximal tubule in superficial rabbit nephrons is similar to the ratio found in other species.

The pathogenesis of ischemic acute kidney injury remains unclear. Red blood cell (RBC) trapping presents as the accumulation of many, tightly packed, RBC in the outer-medullary plexus and is a hallmark of ischemic AKI. We have recently reported that extravasation of toxic heme from the RBC congested medullary capillaries is a cause of tissue injury. This suggests that preventing RBC trapping from occurring may limit tissue injury. The pathogenesis of RBC trapping, however, is poorly understood. While it is clear that RBC trapping occurs in response to occlusion of vessels that drain the renal medulla, the location, as well as the nature of the occlusion, remain unknown. The current study was designed to identify the location of the obstruction responsible for RBC trapping in the rat kidney following ischemia/reperfusion. Our study was performed in 10–12-week-old male and female Sprague Dawley rats. Rats were anesthetized with isoflurane (2-5%) and body temperature maintained at 37°C using a heated table and UV lamp. Flank incisions were performed, and renal ischemia caused by placing a clamp on one of the renal arteries for 45 minutes followed by a 1-hour reperfusion period. The contralateral kidney was used as a non-ischemic control. At the end of the reperfusion period, kidneys were harvested for histological analysis. To identify the location of the vascular obstruction in the kidney, prior to the end of the ischemic period, either 50mg/kg of the plasma protein marker Evans blue or the lipid dye Vybrant DIL (0.12mg) was injected into the tail vein. In non-ischemic control kidneys, both the cortical and medullary vasculature was stained. In contrast, following 1-hour of reperfusion from ischemia, the majority of the cortical glomeruli were unstained, suggesting poor re-flow in this region. In contrast the juxtamedullary glomeruli that supply the medulla with blood were stained, indicating reperfusion of these vessels. In kidneys in which significant RBC trapping occurred, tightly packed RBCs were observed in both the vasa recta bundles and surrounding outer-medullary plexus capillaries. RBC in the vasa recta bundles stained positive, indicating these RBC had entered the kidney on reperfusion. In contrast, most of the RBCs in the capillary plexus were unstained. The interlobular and arcuate veins that drain the outer medulla often contained what appeared to be sludged blood, which was unstained, indicating these vessels had not reperfused. In many cases, however, an internal core of tightly packed stained RBC could be observed within the center of these vessels, indicating that the obstruction had occurred further downstream. In some animals tightly packed, stained RBC could be observed as far downstream as the large interlobar and segmental veins. In these examples, trapped RBC were surrounded by a homogeneous material, devoid of significant structure or cells. Our data indicate that venous obstruction leading to RBC trapping is localized to the large veins of the kidney which drain both the cortex and medulla. The absence of RBC trapping in the cortex is likely due to failure of this region to reperfuse while the obstruction is present. The nature of the obstruction remains unclear. In some cases, unstained sludged blood appeared to line the vascular wall, perhaps preventing smaller veins from draining. In many cases, however, a homogeneous material, largely devoid of cells, appeared to prevent the flow of blood through the large renal veins. Further studies are required to identify the nature of the material obstructing the large veins following ischemia. NIDDK 1R01DK138150-01A1 to Paul O'Connor AHA Transformational Project Award to Paul O'Connor APS SURF fellowship to Angela Ajith This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.