Podocyte Foot Process Effacement Precedes Albuminuria and Glomerular Hypertrophy in CD2-Associated Protein Deficient Mice

Background: Podocyte foot process effacement is a key histologic finding in proteinuric kidney disease. We previously showed that 3-week old CD2AP-deficient mice have significant proteinuria, glomerular hypertrophy and mesangial expansion. The goal of this study is to use morphometry to establish the temporal sequence of podocyte foot process effacement, glomerular volume expansion and albuminuria in Cd2ap−/− mice by measuring these parameters at the 2-week time point.Methods: Wild-type mice age 14 ± 1 days with the Cd2ap gene (WT, N = 5) and mice deficient for Cd2ap (Cd2ap KO, N = 5) were generated. Kidneys were harvested and fixed in 2.5% glutaraldehyde and processed for examination by light and electron microscopy. An average of 415.2 (range 268–716) grid points were counted for all the glomeruli, and quantification of glomerular volume from each kidney. Urine was collected the day prior to sacrifice for urine albumin-to-creatinine ratio (ACR) measurements.Results: There was no difference in albuminuria [median (range) mg/g] between WT [212.2 (177.6–388.4) mg/g] vs. Cd2ap KO mice [203.3 (164.7–910.2) mg/g], P = 0.89; or glomerular volume 68,307[10,931] vs. 66,844[13,022] μm3, p = 0.92. The volume densities of glomerular components of the podocyte, capillary lumen and mesangium were not different for the two groups, P = 0.14, 0.14 and 0.17 respectively. However, foot process width was increased in Cd2ap KO 1128[286] vs. WT [374 ± 42] nm, P = 0.02.Conclusion: Here we show that while 2-week old WT and Cd2ap KO mice have similar levels of albuminuria, glomerular and mesangial volume, Cd2ap KO mice have more extensive podocyte foot process effacement. The data suggests that podocyte injury is the initiating event leading to mesangial expansion and albuminuria in this model.

The Association between Glomerular Diameter and Secondary Focal Segmental Glomerulosclerosis in Chronic Kidney Disease

<b><i>Introduction:</i></b> When nephron loss occurs, the glomerular filtration rate (GFR) is suggested to be maintained by glomerular hypertrophy, but excessive hypertrophy can rather lead to the formation of focal segmental glomerulosclerosis (FSGS), thereby causing progressive kidney damage. However, it is not clear how much glomerular hypertrophy leads to the formation of FSGS. We examined the association between glomerular diameter and FSGS lesions in chronic kidney disease (CKD) patients. <b><i>Methods:</i></b> We recruited 77 patients who underwent renal biopsy during 2016–2017; however, those identified with primary FSGS and glomerulonephritis with active glomerular lesion were excluded. We evaluated the maximal glomerular diameter (Max GD), an indicator of glomerular size, in each renal biopsy specimen and examined its association with FSGS lesion. <b><i>Results:</i></b> The median age, blood pressure, and estimated GFR of the patients were 53 years, 122/70 mm Hg, and 65 mL/min/1.73 m², respectively. The optimal cutoff threshold of Max GD for predicting the presence of FSGS lesions, assessed by receiver operating characteristic curve analysis, was determined to be at 224 μm (area under the curve, 0.81; sensitivity, 81%; specificity, 72%). Multivariate logistic regression analyses demonstrated that Max GD ≥224 μm was significantly associated with the presence of FSGS lesions, independent of other confounding factors (odds ratio, 11.70; 95% confidence interval, 1.93–70.84). <b><i>Discussion/Conclusion:</i></b> Glomerular hypertrophy (Max GD ≥224 μm) has been associated with FSGS lesions in CKD patients and may reflect the limits of the compensatory process.

The Pathological Mechanisms of Obesity-Related Glomerulopathy: A review article

The rising prevalence of obesity-related glomerulopathy (ORG) occurs in accordance with the rising prevalence of obesity worldwide. Clinically ORG is manifested by slowly progressing microalbuminuria that may develop to clinically evident proteinuria. Pathological characteristics of ORG include glomerular hypertrophy in the presence or absence of focal segmental glomerulosclerosis (FSGS). ORG can develop into clinically overt chronic renal insufficiency or even end-stage kidney disease. This article reviews the most important mechanisms for the development of ORG; that are abnormal renal hemodynamics, stimulation of renin-angiotensin-aldosterone system (RAAS), impairment of insulin sensetivity, ectopic lipid deposition, adipose tissue cytokine disorder and local renal micro-inflammation.

MO624COMBINATION TREATMENT WITH LISINOPRIL AND EMPAGLIFLOZIN IMPROVES BIOCHEMICAL AND HISTOLOGICAL MARKERS OF DIABETIC NEPHROPATHY IN HYPERTENSIVE UNINEPHRECTOMIZED DB/DB MICE

Background and Aims Emerging treatments of diabetic kidney disease (DKD) include SGLT2 inhibitors and GLP-1 receptor agonists that are nephroprotective beyond their blood glucose lowering effects. Despite this progress, patients with diabetes are still at risk of developing DKD, and drug discovery remains impeded by the lack of reproducible rodent models exhibiting features of human DKD. To confirm the translatability of an advanced mouse model, we tested standard of care in the setting of type 2 diabetes and hypertension. Method Female db/db mice were injected with a renin-encoding adeno-associated virus construct (reninAAV) to induce hypertension and uninephrectomized (UNx) at 7-8 weeks of age. At 12 weeks of age, daily dosing with vehicle, lisinopril, empagliflozin, or the combination (combo) was initiated. Blood glucose (BG) was measured every third week, while urine albumin-to-creatinine (ACR) and KIM1 were measured in spot urine samples collected before termination at 24 weeks of age. Cystatin C was measured in terminal plasma, while terminal kidney samples were collected for 3D light sheet microscopy and 2D histology: Glomerulosclerosis scoring was performed by AI-assisted automized scoring, whereby glomeruli were given scores from 0 (normal glomerulus) to 4 (glomerulus with global glomerulosclerosis), and morphometric quantification of kidney collagen 3, CD11b, and KIM1 load was performed. Results In reninAAV UNx db/db mice, 12 weeks treatment with empagliflozin and combo reduced fed BG (12.3±1.5 and 13.1±0.9 mM, vehicle: 18.0±1.9 mM) and HbA1c (5.5±0.2 and 5.8±0.2%, vehicle: 7.4±0.03%). Treatment with lisinopril and combo reduced urine ACR (8110±1320 and 2508±346 µg/mg, vehicle: 26,138±1820 µg/mg) and KIM1-to-creatinine (7.5±1.1 and 3.9±0.6 ng/mg, vehicle: 24.8±3.2 ng/mg), while treatment with empagliflozin alone worsened urine ACR (41,523±4670 µg/mg). Glomerular hypertrophy as assessed by 3D imaging was reduced in reninAAV UNx db/db mice combo treated animals compared to vehicle (glomerular volume: 1.46*105±4.3*103 and 1.65*105±4.3*103 µm3), while treatment with empagliflozin alone worsened glomerular hypertrophy (1.83*105 µm3). The total number of glomeruli per kidney was not affected by treatments. Compared to vehicle treatment, lisinopril and combo treatment reduced the fraction of score 3 + 4 glomeruli (% GS 3 + 4 glomeruli: 0.38±0.06 and 0.27±0.03%, vehicle: 0.54±0.05%) Glomerulosclerosis index (GSI) was also reduced by lisinopril and combo treatment (GSI: 2.29±0.10 and 2.04±0.05, vehicle: 2.62±0.09). Treatment with empagliflozin alone worsened GSI (2.95±0.11). Whereas treatments did not significantly affect collagen 3, lisinopril and combo treatment reduced CD11b (total CD11b mass: 0.25±0.03 and 0.24±0.04 mg, vehicle: 0.57±0.07 mg) and KIM1 (total KIM1 mass: 0.25±0.06 and 0.17±0.09 mg, vehicle: 2.01±0.37 mg), whereas empagliflozin did not affect CD11b and KIM1. Conclusion Responses to the combination treatment with lisinopril and empagliflozin showed improvement of urine and histological markers of DKD. Together, these data confirm the translatability of the ReninAAV UNx db/db mouse model of DKD in type 2 diabetes.

LncRNA NEAT1 Promotes High Glucose-Induced Mesangial Cell Hypertrophy by Targeting miR-222-3p/CDKN1B Axis

Glomerular hypertrophy is an early morphological alteration in diabetic nephropathy. Cyclin-Dependent Kinases have been shown to be required for high glucose (HG)-induced hypertrophy; however, the upstream regulators of CDKN1B in glomerular hypertrophy remain unclear. Herein we describe a novel pathway in which Long noncoding RNA (lncRNA) NEAT1 regulates the progression of mesangial cell hypertrophy via a competing endogenous RNA (ceRNA) mechanism. Real-time PCR was performed to detect the relative NEAT1 and miR-222-3p expressions and further confirmed the relationship between NEAT1 and miR-222-3p. Cell cycle was evaluated by flow cytometry. The related mechanisms were explored by Western blot, RNA immunoprecipitation and chromatin immunoprecipitation assay. We show that NEAT1 forms double stranded RNA (dsRNA) with miR-222-3p, thus limiting miR-222-3p’s binding with CDKN1B. This release of CDKN1B mRNA leads to elevated CDKN1B protein expression, resulting in hypertrophy. In addition, we demonstrated that STAT3 which is activated by HG induces the transcription of NEAT1 by binding to its promoter. Our findings underscore an unexpected role of lncRNAs on gene regulation and introduce a new mode of proliferation regulation in mesangial cells.

UJI EFEKTIVITAS EKSTRAK BIJI KETUMBAR (CORIANDRUM SATIVUM L.) TERHADAP GAMBARAN HISTOPATOLOGI GINJAL TIKUS HIPERKOLESTEROLEMIA DIABETES

Diabetes Mellitus (DM) is metabolic disease characterized hyperglicemia caused by abnormalities of insulin secretion, insulin sensitivity, or both of them. Persistent hyperglicemia can trigger the production of oxidative stress. Oxidative stress can cause glomerular hypertrophy. Coriander seed extract has the potential to overcome oxidative stress. This study aims to determine the effect of coriander seed extract to kidney histopathological examination, blood cholesterol, and blood sugar level in hypercholestrolemia diabetic rats. Samples consist of 30 males wistar rats were divided into five treatments i.e: (K1) standard feed and aquades, (K2) high-fat feed, alloxan, and glibenclamid 0,045 mg/day, (K3) high-fat feed, alloxan, and coriander seed extract 300 mg/kgBW/day, (K4) high-fat feed, alloxan, and coriander seed extract 500mg/kgBW/day, (K5) high-fat feed, alloxan, and coriander seed extract 700mg/kgBW/day. Coriander seed extract was given for 28 days. The result of Wilcoxon test showed that coriander seed extract can reduce blood cholesterol level in diabetic hypercholesterolemia, meanwhile the result of paired T-test showed that it can reduce blood sugar level. Measurement of kidney’s histopathological structure as glomerular area was analyzed by Kruskal Wallis test (p=0,001) and continued with Mann-Whitney test. Group K4 can reduce glomerular area significantly compared with group K3 and K5. In conclussion, coriander seed extract can reduce blood cholesterol, blood sugar level and effectively reduce glomerular hypertrophy at dose of 500mg/kgBW/day. Keyword : Coriandrum sativum L.; coriander seed extract; glomerular hypertrophy; blood cholesterol level; blood glucose level ABSTRAK Diabetes Melitus (DM) merupakan suatu penyakit metabolik dengan karakteristik hiperglikemi yang terjadi karena kelainan sekresi insulin, kerja insulin atau kedua-duanya. Hiperglikemi persisten dapat memicu produksi stres oksidatif. Stres oksidatif dapat menyebabkan terjadinya hipertrofi glomerulus. Ekstrak biji ketumbar (Coriandrum sativum L.) berpotensi mengatasi stres oksidatif. Penelitian ini bertujuan untuk mengetahui efek pemberian esktrak biji ketumbar (Coriandrum sativum L.) terhadap gambaran histopatologi ginjal, kadar kolesterol darah, dan kadar gula darah tikus model hiperkolesterolemia diabetes. Sebanyak 30 ekor tikus putih jantan galur wistar, dikelompokkan menjadi lima kelompok dengan perlakuan berbeda yaitu: (K1) pakan standar dan aquades, (K2) pakan tinggi lemak, aloksan, dan glibenklamid 0,045mg/hari, (K3) pakan tinggi lemak, aloksan dan ekstrak biji ketumbar 300mg/kgBB/hari, K4) pakan tinggi lemak, aloksan dan ekstrak biji ketumbar 500mg/kgBB/hari, (K5) pakan tinggi lemak, aloksan, dan ekstrak biji ketumbar 700mg/kgBB/hari. Ekstrak biji ketumbar diberikan selama 28 hari. Hasil uji Wilcoxon menunjukkan ekstrak biji ketumbar dapat menurunkan kadar kolesterol darah, sedangkan hasil uji T berpasangan menunjukkan ekstrak biji ketumbar dapat menurunkan kadar guka darah sewaktu pada tikus model hiperkolesterolemia diabetes. Histopatologi ginjal dianalisis menggunakan uji Kruskal Wallis didapatkan (p=0,001) dan dilanjutkan dengan uji Mann-Whitney. Pada kelompok K4 terdapat penurunan luas glomerulus yang signifikan dibanding kelompok K3 dan K5. Kesimpulannya, esktrak biji ketumbar dapat menurunkan kadar kolesterol darah, kadar gula darah, dan pada dosis 500mg/kgBB/hari paling efektif untuk menurunkan hipertrofi gomerulus.

Mechanistic Insights of Soluble Uric Acid-related Kidney Disease

Hyperuricemia, defined as the presence of elevated serum uric acid (sUA), could lead to urate deposit in joints, tendons, kidney and other tissues. Hyperuricemia as an independent risk factor was common in patients during the causation and progression of kidney disease. Uric acid is a soluble final product of endogenous and dietary purine metabolism, which is freely filtered in kidney glomeruli where approximately 90% of filtered uric acid is reabsorbed. Considerable studies have demonstrated that soluble uric acid was involved in the pathophysiology of renal arteriolopathy, tubule injury, tubulointerstitial fibrosis, as well as glomerular hypertrophy and glomerulosclerosis. In the review, we summarized the mechanistic insights of soluble uric acid related renal diseases.

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy

BackgroundGlomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN).MethodsUsing light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN.ResultsAt 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification.ConclusionsOverall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

Angiotensin blockade attenuates diabetic nephropathy in hypogonadal adult male rats

This study examined the effect of the aromatase inhibitor letrozole (0.5 mg/kg) alone or in combination with the angiotensin-receptor blocker valsartan (30 mg/kg) against streptozocin-induced diabetic nephropathy (DN) in hypogonadal (HG) rats for 12 weeks. First, we tested the HG effect on hormone levels, inflammatory cytokines, and oxidative stress in nondiabetic (ND) and diabetic (D) rats. HG was induced with the luteinizing hormone-releasing hormone antagonist cetrorelix (0.71 mg/kg). Diabetes enhanced hormonal hypogonadism and increased inflammation and oxidative stress. Next, experiments examined the effect of early letrozole and valsartan intervention on DN in HG rats. HG-ND and HG-D rats were treated with letrozole alone or in combination with valsartan. HG-D rats developed proteinuria and had increased blood urea nitrogen and creatinine, and histopathological evidence of renal injury, including glomerular hypertrophy and mesangial expansion. Valsartan alone or in combination with letrozole reduced proteinuria, improved renal functions, and reduced diabetes-induced renal angiotensin II. Both agents ameliorated nuclear factor kappa light chain enhancer of activated B cells, interleukin 1β, interleukin 6, and tumor necrosis factor alpha levels. The combination decreased superoxide dismutase, malondialdehyde, and glutathione peroxidase levels, and prevented glomerular hypertrophy. In HG-D rats, valsartan reduced renal collagen IV and transforming growth factor-beta 1, especially when the testosterone level was corrected by letrozole. Thus, normalizing testosterone and inhibiting renal angiotensin II have a renoprotective effect against DN in HG male rats.