Glomerular Basement Membrane Width Research Articles

Digital pathology assessment of kidney glomerular filtration barrier ultrastructure in an animal model of podocytopathy.

Transmission electron microscopy (TEM) images can visualize kidney glomerular filtration barrier ultrastructure, including the glomerular basement membrane (GBM) and podocyte foot processes (PFP). Podocytopathy is associated with glomerular filtration barrier morphological changes observed experimentally and clinically by measuring GBM or PFP width. However, these measurements are currently performed manually. This limits research on podocytopathy disease mechanisms and therapeutics due to labor intensiveness and inter-operator variability. We developed a deep learning-based digital pathology computational method to measure GBM and PFP width in TEM images from the kidneys of Integrin-Linked Kinase (ILK) podocyte-specific conditional knockout (cKO) mouse, an animal model of podocytopathy, compared to wild-type (WT) control mouse. We obtained TEM images from WT and ILK cKO littermate mice at 4 weeks old. Our automated method was composed of two stages: a U-Net model for GBM segmentation, followed by an image processing algorithm for GBM and PFP width measurement. We evaluated its performance with a 4-fold cross-validation study on WT and ILK cKO mouse kidney pairs. Mean (95% confidence interval) GBM segmentation accuracy, calculated as Jaccard index, was 0.73 (0.70-0.76) for WT and 0.85 (0.83-0.87) for ILK cKO TEM images. Automated and manual GBM width measurements were similar for both WT (p=0.49) and ILK cKO (p=0.06) specimens. While automated and manual PFP width measurements were similar for WT (p=0.89), they differed for ILK cKO (p<0.05) specimens. WT and ILK cKO specimens were morphologically distinguishable by manual GBM (p<0.05) and PFP (p<0.05) width measurements. This phenotypic difference was reflected in the automated GBM (p<0.05) more than PFP (p=0.06) widths. These results suggest that certain automated measurements enabled via deep learning-based digital pathology tools could distinguish healthy kidneys from those with podocytopathy. Our proposed method provides high-throughput, objective morphological analysis and could facilitate podocytopathy research and translate into clinical diagnosis.

Associations of Biomarkers of Tubular Injury and Inflammation with Biopsy Features in Type 1 Diabetes.

Whether biomarkers of tubular injury and inflammation indicate subclinical structural kidney pathology early in type 1 diabetes remains unknown. We investigated associations of biomarkers of tubular injury and inflammation with kidney structural features in 244 adults with type 1 diabetes from the Renin-Angiotensin System Study, a randomized, placebo-controlled trial testing effects of enalapril or losartan on changes in glomerular, tubulointerstitial, and vascular parameters from baseline to 5-year kidney biopsies. Biosamples at biopsy were assessed for kidney injury molecule 1 (KIM-1), soluble TNF receptor 1 (sTNFR1), arginine-to-citrulline ratio in plasma, and uromodulin and epidermal growth factor (EGF) in urine. We examined cross-sectional correlations between biomarkers and biopsy features and baseline biomarker associations with 5-year changes in biopsy features. Participants' mean age was 30 years (SD 10) and diabetes duration 11 years (SD 5); 53% were women. The mean GFR measured by iohexol disappearance was 128 ml/min per 1.73 m 2 (SD 19) and median urinary albumin excretion was 5 μ g/min (interquartile range, 3-8). KIM-1 was associated with most biopsy features: higher mesangial fractional volume (0.5% [95% confidence interval (CI), 0.1 to 0.9] greater per SD KIM-1), glomerular basement membrane (GBM) width (14.2 nm [95% CI, 6.5 to 22.0] thicker), cortical interstitial fractional volume (1.1% [95% CI, 0.6 to 1.6] greater), fractional volume of cortical atrophic tubules (0.6% [95% CI, 0.2 to 0.9] greater), and arteriolar hyalinosis index (0.03 [95% CI, 0.1 to 0.05] higher). sTNFR1 was associated with higher mesangial fractional volume (0.9% [95% CI, 0.5 to 1.3] greater) and GBM width (12.5 nm [95% CI, 4.5 to 20.5] thicker) and lower GBM surface density (0.003 μ m 2 / μ m 3 [95% CI, 0.005 to 0.001] lesser). EGF and arginine-to-citrulline ratio correlated with severity of glomerular and tubulointerstitial features. Baseline sTNFR1, uromodulin, and EGF concentrations were associated with 5-year glomerular and tubulointerstitial feature progression. Biomarkers of tubular injury and inflammation were associated with kidney structural parameters in early type 1 diabetes and may be indicators of kidney disease risk. Renin Angiotensin System Study (RASS/B-RASS), NCT00143949. This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_11_17_CJN0000000000000333.mp3.

Molecular programs associated with glomerular hyperfiltration in early diabetic kidney disease

Hyperfiltration is a state of high glomerular filtration rate (GFR) observed in early diabetes that damages glomeruli, resulting in an iterative process of increasing filtration load on fewer and fewer remaining functional glomeruli. To delineate underlying cellular mechanisms of damage associated with hyperfiltration, transcriptional profiles of kidney biopsies from Pima Indians with type 2 diabetes with or without early-stage diabetic kidney disease were grouped into two hyperfiltration categories based on annual iothalamate GFR measurements. Twenty-six participants with a peak GFR measurement within two years of biopsy were categorized as the hyperfiltration group, and 26 in whom biopsy preceded peak GFR by over two years were considered pre-hyperfiltration. The hyperfiltration group had higher hemoglobin A1c, higher urine albumin-to-creatinine ratio, increased glomerular basement membrane width and lower podocyte density compared to the pre-hyperfiltration group. A glomerular 1240-gene transcriptional signature identified in the hyperfiltration group was enriched for endothelial stress response signaling genes, including endothelin-1, tec-kinase and transforming growth factor-β1 pathways, with the majority of the transcripts mapped to endothelial and inflammatory cell clusters in kidney single cell transcriptional data. Thus, our analysis reveals molecular pathomechanisms associated with hyperfiltration in early diabetic kidney disease involving putative ligand-receptor pairs with downstream intracellular targets linked to cellular crosstalk between endothelial and mesangial cells.

398-P: KIM-1 and TNFR 1 and Associations with Early Diabetic Nephropathy Histology

Aim: GFR and urine albumin excretion (UAE) are prevailing measures used to detect and monitor diabetic nephropathy (DN) . We tested whether serum kidney injury molecule-1 (KIM-1) and tumor necrosis factor receptor-1 (TNFR1) , markers of DN progression, are associated with DN renal structural parameters in persons with TIDM. Methods: We measured KIM-1 and TFNR1 in 285 participants from the Renin Angiotensin System Study (RASS) . Participants had T1DM for 2-20 yrs and were free of any clinical DN. Baseline iGFR and kidney biopsies were obtained. We used linear regression to estimate cross-sectional associations of markers with renal structural measures. Results: Mean age was 30 ± yrs; mean iGFR was 125 ± 18 ml/min/1.73 m2; median UAE was 5 (IQR 4- 7) . Higher KIM-1 was associated with significantly greater mesangial fraction volume [Vv (Mes/glom) ] and the tubular atrophy (table 1) . Higher TNFR1 was associated with significantly higher Vv (Mes/glom) and glomerular basement membrane (GBM) width. Neither iGFR nor UAE were significantly associated with lesions. Conclusion: Serum KIM-1 and TNFR1 were associated with higher values for Vv (Mes/glom) , GBM, and tubular atrophy in persons with T1DM and normal kidney function, suggesting relevance as markers of early DN structural changes prior to UAE elevation or GFR decline. KIM-1 is specific to the tubules, highlighting the importance of characterizing the natural history of tubular functions in DN. Disclosure F.Tian: None. D.Prince: None. A.Doria: Research Support; Novo Nordisk Foundation. M.Mauer: None. B.Kestenbaum: Consultant; Reata Pharmaceuticals, Inc. Funding T32

MO399: Remodel: A Mechanistic Trial Evaluating the Effects of Semaglutide on the Kidneys In People With Type 2 Diabetes and Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Approximately 40% of people with type 2 diabetes (T2D) develop chronic kidney disease (CKD) and, despite current treatment, T2D is the most common cause of progression to kidney failure. This situation underscores the need for additional pharmacotherapeutic options. Analyses of cardiovascular outcomes trials suggest that glucagon-like peptide-1 receptor agonists (GLP-1RAs), such as semaglutide, lower albuminuria and attenuate estimated glomerular filtration rate (eGFR) decline in people with T2D. Previous analyses suggest that GLP-1RAs reduce hypoxia and inflammation and, thereby, have a mode of action on the kidneys that is distinct from other treatments, such as sodium-glucose cotransporter-2 (SGCT-2) inhibitors and renin–angiotensin–aldosterone system (RAAS) blockers. Furthermore, the primary benefit of semaglutide appears to be in people with an eGFR &amp;lt; 60 mL/min/1.73 m2, a group in which there is a significant residual risk of progression and a consequent unmet need for effective treatment. To gain further insights on the kidney-protective mechanism of action of semaglutide, the REMODEL trial is integrating investigative functional kidney magnetic resonance imaging (MRI) and kidney biopsies; recent developments in these techniques permit elucidation of the mechanisms underlying kidney protection with current therapies. Mechanistic findings of the REMODEL trial will complement those of the ongoing FLOW clinical trial, which is designed to evaluate clinical outcomes in people with T2D and CKD treated with once-weekly (OW) subcutaneous semaglutide. METHOD REMODEL (NCT04865770) is a 52-week, multicentre, international clinical trial (Figure 1). Primary endpoints are MRI-based and include change from baseline to week 52 in kidney oxygenation (measured with BOLD MRI R2*), global kidney perfusion (phase-contrast MRI) and kidney inflammation (T1 Mapping MRI). Secondary endpoints evaluated from kidney biopsies in a nested cohort (n ∼ 45) include change from baseline to week 52 in intrarenal mRNA expression, assessed by single-nucleus transcriptomics and glomerular basement membrane width, assessed by morphometry. Other secondary endpoints include the apparent diffusion coefficient (estimating renal fibrosis; evaluated with diffusion-weighted MRI), natriuresis, albumin excretion rate and creatinine clearance. MRI outcomes will also be evaluated at week 4 to identify potential early effects of semaglutide in the kidney. Examples of MRI and kidney biopsy single-cell gene expression profile data from participants with CKD are shown in Figure 2 (data not from REMODEL). Safety will be assessed throughout the trial. RESULTS REMODEL was initiated in April 2021 and is being conducted in Canada, France, Italy, Poland, South Africa, Spain and USA. CONCLUSION REMODEL will investigate the effect of the GLP-1RA semaglutide on inflammatory and hypoxia-related pathways in the kidney. The combination of MRI and tissue-level interrogation with biopsies will complement standard laboratory findings, enabling the identification of cells and pathways involved in kidney disease and protection. The trial will provide valuable mechanistic insights on the use of OW semaglutide in people with T2D and CKD and may stimulate the development of a precision medicine approach to the management of such individuals. In addition, REMODEL will complement the findings of the FLOW trial.

MO255: Sparsentan Protects the Glomerular Basement Membrane and Glycocalyx, and Attenuates Proteinuria in a Rat Model of Focal Segmental Glomerulosclerosis (FSGS)

Abstract BACKGROUND AND AIMS Sparsentan is a novel, dual acting, highly selective antagonist of the endothelin (ET-1) type A receptor (ETAR) and the angiotensin II (Ang II) subtype 1 receptor (AT1R). This first in class dual endothelin angiotensin receptor antagonist (DEARA) is being investigated in phase 3 clinical trials for focal segmental glomerulosclerosis (FSGS) and IgA nephropathy. Considering the broad spectrum of renal actions of ET-1 and Ang II, inhibition of both pathways using sparsentan is postulated to target multiple renal cell types and thereby ameliorate multiple pathophysiologic aspects of glomerular disease. The Adriamycin-induced nephropathy (ADR) rat model is a well-established model characterized by rapid podocyte injury and proteinuria followed by glomerulosclerosis (GS), with lesions reflective of those in human FSGS. The ADR rat model was used to assess the ability of sparsentan to attenuate kidney injury in an experimental FSGS setting. METHOD ADR was induced by a single IV dose of Adriamycin to male Sprague Dawley rats. Sham control animals (n = 10) received the Adriamycin vehicle only. Following disease induction, rats (10/group) were administered 0.5% MC/0.25% Tween 80 vehicle (VEH) or sparsentan at 20 (Spar20), 60 (Spar60), or 180 mg/kg (Spar180) by oral gavage once daily for 35 days and underwent periodic measurements of proteinuria (urine protein: creatinine ratio; UPCR). At the end of the treatment period, histological evaluations of the kidney were made by light and electron microscopy, and by immunohistochemistry to determine GS score (GSS, glomerular staining as %field area), podocyte number (p57 positive nuclei, count per glomerular area), glomerular basement membrane (GBM) morphology, glomerular glycocalyx (GLX) integrity (colloidal iron), and inflammatory cell infiltration (ED1 positivity, area of glomerular staining as %field area). RESULTS Numeric data are presented as mean ± SD. In groups dosed with Spar60 or Spar180 compared with VEH: the development of GSS was significantly reduced (P &amp;lt; .0001; Spar60, 1.14 ± 0.21 or Spar180, 1.23 ± 0.21 versus VEH, 2.70 ± 0.14), increase in GBM width was significantly attenuated (Fig. 1), GLX staining was significantly greater (P = .0079, Spar60, 18.6 ± 1.86 or P = 0.0112, Spar180, 17.6 ± 2.40 versus VEH, 10 ± 1.18), and inflammatory cell infiltrate was significantly reduced (P = .0058, Spar60, 1.5 ± 0.43 or P = .0004, Spar180, 1.1 ± 0.16 versus VEH, 4.1 ± 0.66) in sparsentan-treated rats as compared with VEH. Additionally, podocyte number was significantly higher in the Spar180 group compared with VEH (P = .004, Spar180, 6.9 ± 0.91 versus VEH, 4.1 ± 0.31). Sparsentan-treated groups showed a consistent trend toward preservation of podocyte foot process width and number compared with VEH (Fig. 1). Further, Spar180 significantly attenuated development of UPCR at Day 14 compared with VEH (P = .0033) and there was a trend toward dose-dependent attenuation on both Day 14 and Day 33. CONCLUSION Dual antagonism of ETAR and AT1R by sparsentan protected rat kidneys in the ADR model of FSGS, as measured by its impact on multiple pathological disease features of the model, including attenuation of UPCR and podocyte loss, maintenance of GBM width, protection of GLX and reduction in glomerular macrophage infiltration.

Structural Lesions on Kidney Biopsy in Youth-Onset and Adult-Onset Type 2 Diabetes.

Type 2 diabetes (T2D) is a leading cause of end-stage kidney disease worldwide. Recent studies suggest a more aggressive clinical course of diabetic kidney disease in youth-onset compared with adult-onset T2D. We compared kidney structural lesions in youth- and adult-onset T2D to determine if youth onset was associated with greater early tissue injury. Quantitative microscopy was performed on kidney tissue obtained from research kidney biopsies in 161 Pima Indians (117 women, 44 men) with T2D. Onset of T2D was established by serial oral glucose tolerance testing, and participants were stratified as youth onset (age <25 years) or adult onset (age ≥25 years). Associations between clinical and morphometric parameters and age at onset were tested using linear models. At biopsy, the 52 participants with youth-onset T2D were younger than the 109 with adult-onset T2D (39.1 ± 9.9 vs. 51.4 ± 10.2 years; P < 0.0001), but their diabetes duration was similar (19.3 ± 8.1 vs. 17.0 ± 7.8 years; P = 0.09). Median urine albumin-to-creatinine ratio was higher in the youth-onset group (58 [25th-75th percentile 17-470] vs. 27 [13-73] mg/g; P = 0.02). Youth-onset participants had greater glomerular basement membrane (GBM) width (552 ± 128 vs. 490 ± 114 nm; P = 0.002) and mesangial fractional volume (0.31 ± 0.10 vs. 0.27 ± 0.08; P = 0.001) than adult-onset participants. Glomerular sclerosis percentage, glomerular volume, mesangial fractional volume, and GBM width were also inversely associated with age at diabetes onset as a continuous variable. Younger age at T2D onset strongly associates with more severe kidney structural lesions. Studies are underway to elucidate the pathways underlying these associations.

Serum Level of Polyubiquitinated PTEN and Loss of Kidney Function in American Indians With Type 2 Diabetes

Fibrosis is a major driverof chronic kidney disease, and epithelial-mesenchymal transition (EMT) may contribute to its development. A polyubiquitinated form ofphosphatase and tensin homolog (PTENK27polyUb) promotes EMT invitro. Thus, it is a potentially useful biomarker of progressive kidney fibrosis and may predict loss of kidney function. Observational cohort study. Southwest United States, American Indians (154 women, 80 men) with or at high risk for diabetic kidney disease (DKD). Serum level of PTENK27polyUb. ≥40% loss of glomerular filtration rate (GFR) or onset of kidney failure. Kidney structural measures in a subset of study participants who underwent research kidney biopsies (n= 77). Cox proportional hazards models adjusted for age, sex, diabetes duration, hemoglobin A1c (HbA1c), blood pressure, use of renin angiotensin system (RAS) blockers, measured GFR, and albuminuria. Spearman correlations for associations with structural measures. At baseline, the participants' mean age was 42.8 ± 10.5 (SD) years, diabetes duration 11.5 ± 7.1 years, mean arterial pressure 90.5 ± 9.5 mm Hg, HbA1c 9.3 ± 2.4%, GFR 152 ± 45 mL/min, and median urinary albumin-creatinine ratio 38 (interquartile range, 14-215) mg/g. RAS blockers were being used by 64 participants (27.4%). A higher PTENK27polyUb value was associated with a greater risk of≥40% loss of GFR during a median follow-up period of 6.3 years (HR for quartile 4 [Q4] vs Q1, 3.95 [95% CI, 2.23-6.98], P< 0.001). Serum PTENK27polyUb was associated with an increased risk of kidney failure over a median follow-up period of 15.8 years (HR for Q4 vs Q1, 5.66 [95% CI, 1.99-16.13], P= 0.001). Baseline serum PTENK27polyUb in the biopsy subset correlated with structural measures including glomerular basement membrane width (ρ= 0.370, P< 0.001) and mesangial fractional volume (ρ= 0.392, P< 0.001). Small study in single population. Higher serum PTENK27polyUb is associated with increased risk for GFR decline and kidney failure in American Indians with type 2 diabetes.

Quality assurance: Evaluation and comparison of methods for electron microscopic measurement of GBM width and the effect of in-lab calibration in diagnostic renal pathology.

Replacing equipment and software can improve efficiency and allow updates to laboratory procedures, but has the potential to introduce changes in established values for a laboratory. Replacement of an electron microscope (EM), fitted with an updated digital camera, and use of new software for imaging and analysis prompted this QA study to ensure that new equipment, imaging, and measurement of the glomerular basement membrane (GBM) produced data consistent with the laboratory's established range of normal width. GBM measurements from 14 randomly selected human renal biopsies were compared using five different approaches. Original measurements of GBMs obtained on the laboratory's previous EM were compared to images collected on the new microscope with measurements performed using new software, as well as the original images and the new images measured using a separate software method as a control. The widths obtained by five approaches were compared to each other. While measurements showed minor variability between the approaches, significant difference in GBM width was noted in three of the paired comparisons. In some cases, these differences suggested slight diagnostic changes. Evaluation of new equipment, software, and techniques is important for a laboratory's quality assurance. While new equipment and/or procedures can introduce errors in test outcomes, we found that different EMs, cameras, and software made slight differences in our laboratory's values for kidney GBM width. However, a few cases showed enough difference in GBM width to suggest a change in diagnosis, illustrating the necessity of calibration adjustments in the setting of new equipment and software.

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Saxagliptin attenuates glomerular podocyte injury by increasing the expression of renal nephrin and podocin in type 2 diabetic rats.

To observe the effects of saxagliptin on the expression of mitogen-activated protein kinase 38 (p38MAPK), nephrin and podocin in renal tissue in type 2 diabetic (T2DM) rats, and to explore the possible mechanism of its renal protection. Forty-eight male Sprague-Dawley rats were used for the study and divided into four different groups: normal controls (Group NC), DM controls (Group DM), DM + glibenclamide (Group Su) and DM + saxagliptin (Group Sa). The day drug administration started was defined as week 0. After 12weeks, hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG), urea nitrogen (BUN) and creatinine (Cr) in serum were detected, simultaneously albumin and creatinine in urine were measured, respectively, and then urinary albumin/creatinine ratio (UACR) was calculated. The pathological morphology of kidney tissue in different groups was observed, and the expression of nephrin and podocin mRNA and protein in kidney tissue were detected. (1) After 12weeks, FBG and HbA1c in Group Su and Group Sa were significantly lower than those in Group DM (both P < 0.05), while there was no significant difference between Group Su and Group Sa. TC, TG and UACR in Group Sa were significantly decreased thanthose in Group DM. (2) When compared with Group DM, the kidney weight/body weight ratios, the average width of glomerular basement membrane and foot process fusion ratio were all improved in Group Sa after 12weeks. (3) The expression of p38MAPK mRNA and protein was significantly decreased, while nephrin and podocin mRNA and protein were significantly higher in Group Sa than those in Group DM after 12weeks. (4) A significant negative correlation was detected between p38MAPK mRNA and nephrin (r = - 0.421, P = 0.009) and podocin mRNA (r = - 0.570, P = 0.000), respectively. Saxagliptin can reduce urinary albumin excretion and exert renal protective effect, especially on podocytes in T2DM rats. The mechanism may be related to its inhibition of renal p38MAPK signaling pathway and the increase in the expression of nephrin and podocin in renal tissue, which is independent of its hypoglycemic effect.

Empagliflozin and Linagliptin Alleviate Glomerular Changes in a Model of Diabetic Nephropathy

The aim: to assess the effects of SGLT2 inhibitor empagliflozin, DPP-4 inhibitor linagliptin, and their combination on structural changes in kidneys in a model of type 2 diabetic nephropathy. Eight-week-old male db/db diabetic mice (BKS.Cg-Dock7m+/+Leprdb/J) were treated with empagliflozine (10 mg/kg), linagliptin (10 mg/kg), combination of both agents, or placebo for 8 weeks. Nondiabetic heterozygous db/+ mice were used as control. Renal structural changes were analyzed quantitatively from the light and electron microscopic images. Both empagliflozin and linagliptin, either alone or in combination, attenuated mesangial expansion estimated by mesangial volume (p=0.00for empagliflozin, p=0.03 for linagliptin and combination). The width of glomerular basement membrane was diminished in all treated groups (all p&amp;lt;0.01). In diabetic mice, podocytopathy was manifested by the foot process effacement (p=0.03). Under treatment, the number of podocyte foot processes increased (p=0.003 for linagliptin, p=0.03 for empagliflozin and combination) and the width of podocyte foot processes decreased in all treated groups (p=0.003 for empagliflozin and linagliptin, p=0.001 for combintion). The number of endothelial fenestrae in glomerular capillaries was increased after the treatment (all p&amp;lt;0.01). The interaction between empagliflozin and linagliptin was significant for the number and width of podocyte foot processes, and the number of endothelial fenestrae (all p&amp;lt;0.01). The effects of both agents on structural changes were not associated with hypoglycemic activity. The obtained results demonstrate that empagliflozin and linagliptin ameliorate renal fibrosis and podocyte injury in a model of type 2 diabetic nephropathy. Disclosure A.I. Korbut: None. V. Klimontov: None. I. Taskaeva: None. N.P. Bgatova: None. I. Ishchenko: None. E.L. Zavjalov: None.

The relationship between the thickness of glomerular basement membrane and renal outcomes in patients with diabetic nephropathy

Glomerular basement membrane (GBM) thickening is considered as one of the earliest detectable pathological features of diabetic nephropathy (DN). However, whether the thickness of GBM will impact the prognosis of DN remains largely unknown. Our aim was to explore the relationship between thickness of GBM and DN progression in patients with type 2 diabetes mellitus (T2DM). A total of 118 patients with T2DM and biopsy-proven DN who received follow-up for at least 1year were recruited. The patients were divided into two groups according to the median (787nm) of the GBM thickness: Group 1: GBM thickness < 787nm (n = 59), and Group 2: GBM thickness ≥ 787nm (n = 59). The GBM width was estimated by the direct GBM measurements as recently modified by Haas. Renal outcomes were defined by progression to ESRD and/or doubling of serum creatinine (D-Cr). The influence of GBM thickness on renal outcomes was assessed using Cox regression. Compared with the Group 1, patients in Group 2 had more serious renal insufficiency and glomerular lesions. During the follow-up, ESRD occurred in 39.8% of patients, and 8.5% of patients progressed to D-Cr. The univariate analysis indicated the greater width of GBM the higher risk of renal outcomes in T2DM patients with DN (HR [95% CI] = 2.180 [1.246-3.814], p = 0.006). However, the multivariate COX analysis demonstrated that the GBM thickness was not an independent risk factor for progression to ESRD or D-Cr (HR [95% CI] = 0.825 [0.404-1.685], p = 0.597) when adjusting for important clinical variables and pathological findings. In conclusion, the DN patients with greater width of GBM had relatively poorer renal prognosis, although it did not emerge as an independent indicator of disease progression.

Role of Kidney Biopsies for Biomarker Discovery in Diabetic Kidney Disease.

Although estimated glomerular filtration rate and albuminuria are well-established biomarkers of diabetic kidney disease (DKD), additional biomarkers are needed, especially for the early stages of the disease when both albuminuria and estimated glomerular filtration rate may still be in the normal range and are less helpful for identifying those at risk of progression. Traditional biomarker studies for early DKD are challenging because of a lack of good early clinical end points, and most rely on changes in existing imprecise biomarkers to assess the value of new biomarkers. There are well-characterized changes in kidney structure, however, that are highly correlated with kidney function, always precede the clinical findings of DKD and, at preclinical stages, predict DKD progression. These structural parameters may thus serve as clinically useful end points for identifying new biomarkers of early DKD. In addition, investigators are analyzing tissue transcriptomic data to identify pathways involved in early DKD which may have associated candidate biomarkers measurable in blood or urine, and differentially expressed microRNAs and epigenetic modifications in kidney tissue are beginning to yield important observations which may be useful in identifying new clinically useful biomarkers. This review examines the emerging literature on the use of kidney tissue in biomarker discovery in DKD.

Type 1 diabetes mellitus induces structural changes and molecular remodelling in the rat kidney.

There is much evidence that diabetes mellitus (DM)-induced hyperglycemia (HG) is responsible for kidney failure or nephropathy leading to cardiovascular complications. Cellular and molecular mechanism(s) whereby DM can damage the kidney is still not fully understood. This study investigated the effect of streptozotocin (STZ)-induced diabetes (T1DM) on the structure and associated molecular alterations of the isolated rat left kidney following 2 and 4months of the disorder compared to the respective age-matched controls. The results revealed hypertrophy and general disorganized architecture of the kidney characterized by expansion in glomerular borders, tubular atrophy and increased vacuolization of renal tubular epithelial cells in the diabetic groups compared to controls. Electron microscopic analysis revealed ultrastructural alterations in the left kidney highlighted by an increase in glomerular basement membrane width. In addition, increased caspase-3 immunoreactivity was observed in the kidney of T1DM animals compared to age-matched controls. These structural changes were associated with elevated extracellular matrix (ECM) deposition and consequently, altered gene expression profile of ECM key components, together with elevated levels of key mediators (MMP9, integrin 5α, TIMP4, CTGF, vimentin) and reduced expressions of Cx43 and MMP2 of the ECM. Marked hypertrophy of the kidney was highlighted by increased atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression. These changes also correlated with increased TGFβ1 activity, gene expression in the left kidney and elevated active TGFβ1 in the plasma of T1DM rats compared to control. The results clearly demonstrated that TIDM could elicit severe structural changes and alteration in biochemical markers (remodelling) in the kidney leading to diabetic nephropathy (DN).

White blood cell fractions correlate with lesions of diabetic kidney disease and predict loss of kidney function in Type 2 diabetes.

Inflammation linked to diabetic kidney disease (DKD) may affect white blood cell (WBC) counts and differentials. We examined the cross-sectional associations of total WBC count and WBC fractions with structural lesions of DKD in 108 Pima Indians with Type 2 diabetes who underwent research kidney biopsies. We also examined the longitudinal association of these WBC variables with renal function loss (RFL) in 941 Europeans with Type 2 diabetes from the SURDIAGENE study. Associations of WBC variables with morphometric parameters were assessed by linear regression. RFL was defined as≥40% loss of estimated glomerular filtration rate from baseline. Associations with RFL were evaluated by Cox regression. Hazard ratios (HRs) were reported per standard deviation increment of each WBC variable. After multivariable adjustment, lymphocyte (r = -0.20, P = 0.043) and eosinophil (r = 0.21, P = 0.032) fractions in the Pima Indians correlated with glomerular basement membrane width. Eosinophil fraction also correlated with glomerular filtration surface density (r = -0.21, P = 0.031). Lymphocyte fraction (r = 0.25, P = 0.013), neutrophil fraction (r = -0.23, P = 0.021) and the neutrophil:lymphocyte ratio (r = -0.22, P = 0.024) correlated with percentage of normally fenestrated endothelial cells. During median follow-up of 4.5 years, 321 SURDIAGENE participants developed RFL. Lower lymphocyte fraction [HR = 0.67, 95% confidence interval (95% CI) 0.60-0.76] and higher neutrophil fraction (HR = 1.35, 95% CI 1.20-1.52), total WBC count (HR = 1.20, 95% CI 1.08-1.35) and neutrophil:lymphocyte ratio (HR = 1.44, 95% CI 1.28-1.62) each predicted RFL in this cohort. WBC fractions associate with morphometric lesions of DKD and predict RFL in individuals with Type 2 diabetes.

VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.

Key points Progressive depletion of all vascular endothelial growth factor A (VEGF‐A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over‐expression of VEGF‐A165b only.VEGF‐A165b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub‐podocyte space coverage, produced by VEGF‐A depletion.VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference.VEGF‐A165b has opposite effects to VEGF‐A165 on the expression of genes involved in endothelial cell migration and proliferation. Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF‐A). However, little is known about the contribution of VEGF‐A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF‐A165b (resulting from alternative usage of a 3′ splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad‐transgenic model, that over‐expression of VEGF‐A165b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF‐A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub‐podocyte space coverage is reduced when VEGF‐A is depleted, all of which are rescued in VEGF‐A165b over‐expressors. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule‐1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down‐regulates genes involved in migration and proliferation of endothelial cells, otherwise up‐regulated by the canonical isoform VEGF‐A165. The results of the present study indicate that manipulation of VEGF‐A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

The Effects of Extensive Glomerular Filtration of Thin Graphene Oxide Sheets on Kidney Physiology.

Understanding how two-dimensional (2D) nanomaterials interact with the biological milieu is fundamental for their development toward biomedical applications. When thin, individualized graphene oxide (GO) sheets were administered intravenously in mice, extensive urinary excretion was observed, indicating rapid transit across the glomerular filtration barrier (GFB). A detailed analysis of kidney function, histopathology, and ultrastructure was performed, along with the in vitro responses of two highly specialized GFB cells (glomerular endothelial cells and podocytes) following exposure to GO. We investigated whether these cells preserved their unique barrier function at doses 100 times greater than the dose expected to reach the GFB in vivo. Both serum and urine analyses revealed that there was no impairment of kidney function up to 1 month after injection of GO at escalating doses. Histological examination suggested no damage to the glomerular and tubular regions of the kidneys. Ultrastructural analysis by transmission electron microscopy showed absence of damage, with no change in the size of podocyte slits, endothelial cell fenestra, or the glomerular basement membrane width. The endothelial and podocyte cell cultures regained their full barrier function after >48 h of GO exposure, and cellular uptake was significant in both cell types after 24 h. This study provided a previously unreported understanding of the interaction between thin GO sheets with different components of the GFB in vitro and in vivo to highlight that the glomerular excretion of significant amounts of GO did not induce any signs of acute nephrotoxicity or glomerular barrier dysfunction.

Podocyte Detachment Is Associated with Renal Prognosis in ANCA-Associated Glomerulonephritis: A Retrospective Cohort Study.

The prognosis of antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (ANCA-GN) is unfavorable despite immunosuppressive therapy. It has been suggested that the loss of podocytes is a hallmark of progressive kidney disease. However, it is unclear about podocyte injuries and their predictive values on the prognosis in ANCA-GN. Therefore, the current study aimed to investigate the podocyte injury in renal histopathology and its association with renal prognosis of patients with ANCA-GN.A total of 170 patients with ANCA-GN were recruited in this study. Morphometric investigation of podocytes by electron microscopy including foot process width (FPW), podocyte density per glomerulus (Nv), and glomerular basement membrane (GBM) width were measured and calculated in ANCA-GN patients. Cox regression analysis was used to analyze the association between podocyte injuries and prognosis of patients with ANCA-GN.Foot processes broadening, podocyte detachment, and GBM thickening could be observed in electron micrographs in the specimens of 158/170 (92.9%), 142/170 (83.5%), and 150/170 (88.2%) patients, respectively. Compared with normal controls, FPW and GBM width in ANCA-GN patients was significantly higher (1269.39 ± 680.19 vs 585.81 ± 77.16, P = 0.004; 668.23 ± 208.73 vs 354.23 ± 52.70, P = 0.000, respectively), while the podocyte density was significantly lower (55.90 ± 36.32 vs 255.23 ± 47.29, P = 0.000). The podocyte density was independently associated with the recovery of renal function in logistic regression analysis (OR, 1.083; 95% CI, 1.025–1.440; P = 0.005). Furthermore, multivariate analysis revealed that podocyte density was an independent predictor of end-stage renal disease (ESRD) (model A: HR, 0.950; 95% CI, 0.919–1.982; P = 0.002; model B: HR, 0.953; 95% CI, 0.922–0.985; P = 0.004).Podocyte structural damage and detachment occurred frequently in patients with ANCA-GN. Moreover, podocyte detachment was an independent predictor of renal outcomes.

Cardiovascular autonomic neuropathy associates with nephropathy lesions in American Indians with type 2 diabetes

AimsCardiovascular autonomic neuropathy (CAN) predicts clinical diabetic nephropathy (DN). We investigated the relationship between DN structural lesions and CAN. MethodsSixty three Pima Indians with type 2 diabetes underwent kidney biopsies following a 6-year clinical trial testing the renoprotective efficacy of losartan vs. placebo. CAN was assessed a median 9.2years later. CAN variables included expiration/inspiration ratio (E/I), standard deviation of the normal R-R interval (sdNN), and low and high frequency signal power and their ratio (LF, HF, LF/HF); lower values reflect more severe neuropathy. Associations of CAN with renal structural variables were assessed by linear regression adjusted for age, sex, diabetes duration, blood pressure, HbA1c, glomerular filtration rate, and treatment assignment during the trial. ResultsGlobal glomerular sclerosis was negatively associated with sdNN (partial r=−0.35, p=0.01) and LF (r=−0.32, p=0.02); glomerular basement membrane width was negatively associated with all measures of CAN except for LF/HF (r=−0.28 to −0.42, p<0.05); filtration surface density was positively associated with sdNN, LF, and HF (r=0.31 to 0.38, p<0.05); and cortical interstitial fractional volume was negatively associated with HF (r=−0.27, p=0.04). ConclusionsCAN associates with DN lesions.