Basement Membrane Width Research Articles

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.

Effects of Obesity in Old Age on the Basement Membrane of Skeletal Muscle in Mice.

Obesity and aging are known to affect the skeletal muscles. Obesity in old age may result in a poor basement membrane (BM) construction response, which serves to protect the skeletal muscle, thus making the skeletal muscle more vulnerable. In this study, older and young male C57BL/6J mice were divided into two groups, each fed a high-fat or regular diet for eight weeks. A high-fat diet decreased the relative gastrocnemius muscle weight in both age groups, and obesity and aging individually result in a decline in muscle function. Immunoreactivity of collagen IV, the main component of BM, BM width, and BM-synthetic factor expression in young mice on a high-fat diet were higher than that in young mice on a regular diet, whereas such changes were minimal in obese older mice. Furthermore, the number of central nuclei fibers in obese older mice was higher than in old mice fed a regular diet and young mice fed a high-fat diet. These results suggest that obesity at a young age promotes skeletal muscle BM formation in response to weight gain. In contrast, this response is less pronounced in old age, suggesting that obesity in old age may lead to muscle fragility.

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 < 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.

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.

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.

Ultrastructural examination of basement membrane pathology in horses with insulin-induced laminitis

The third phalanx of the equine digit is suspended within the hoof capsule by a specialized interdigitating dermoepidermal layer called the lamellae, which fails during laminitis. Pathology of the basement membrane (BM), which interfaces epidermis and dermis, is evident during acute laminitis. However, BM damage appears to be less prevalent in ponies with the insulin-associated form of laminitis. The aim of the present study was to investigate changes to the ultrastructure and morphometry of the lamellar BM in the acute phase of insulin-induced laminitis in horses. Lamellar tissue from the left forefoot of 3 horses with acute hyperinsulinemic laminitis was examined with transmission electron microscopy and compared with tissue from normal horses. Lamellar BM width and hemidesmosome (HD) density were assessed every 5 μm along ∼200 μm of secondary epidermal lamellar BM. The BM zone of treated horses was extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, fragmentation and disorientation of HDs, and cytoskeletal disengagement of the HDs. The mean (±SD) lamellar BM was twice as wide in treated (0.25 ± 0.05 μm), compared with control (0.14 ± 0.02 μm), horses. The HD density (HDs/μm) was reduced by half in the treatment group (1.88 ± 0.37), compared with controls (3.6 ± 0.13). The reduced number of HDs in horses with laminitis may contribute to the weakening of the dermoepidermal junction and lamellar failure. Disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia, may account for HD loss. Further investigation of the underlying etiopathogenesis of BM dysfunction during hyperinsulinemic laminitis in horses may facilitate an improved understanding of the disease.

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.

Inhibition of Nitric Oxide Synthase (NOS) by NG‐monomethyl‐L‐arginine (L‐NMMA) Reduces Transient Increase in the Blood‐Brain Barrier Solute Permeability in Rat Brain by Transcranial Direct Current Stimulation

Transcranial Direct Current Stimulation (tDCS) is a non‐invasive electrical stimulation technique investigated for a broad range of medical and performance indications. Our prior study has shown that tDCS with proper dosage can achieve non‐invasive, selective and temporary increase in the blood‐brain barrier (BBB) permeability (P). To further investigate the cellular and structural mechanisms by which tDCS increases P, prior to 20 min 1 mA tDCS, we pretreated the cerebral microvessels for ~30 min with a NOS inhibitor, L‐NMMA, through the tail vein injection, at an initial blood concentration of ~1 mM. We then used laser scanning multiphoton microscopy with 840 nm excitation wavelength to determine P of the cerebral microvessels in the rat brain tissue 100–200 μm below the pia mater. Sodium fluorescein (MW 376), or FITC‐dextran 70k in 1% BSA mammalian Ringer was injected into the rat (SD, 250–300g) cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ~3 ml/min. P were determined from the rate of dye spreading images around individual microvessels in the brain tissue. It was found that L‐NMMA pretreatment significantly reduced the increased P to sodium fluorescein from ~13‐fold to ~4‐fold of its control, and P to Dextran‐70k from ~88‐fold to ~8‐fold of its control at 5 min post tDCS treatment. Treatment by L‐NMMA alone for ~55 min significantly decreased the P to both solutes to ~66% of their controls. The predictions from our previously developed mathematical model for the BBB suggested that increasing the cleft width between adjacent endothelial cells and the width of basement membrane between the endothelium and astrocyte foot processes, due to contracting of endothelial cells, should be responsible for the transient increase in P by tDCS. In contrast, pretreatment of L‐NMMA may strengthen the structural components, most likely the junction strands in the cleft between endothelial cells to prevent the permeability increase by tDCS.Support or Funding InformationNIH RO1 NS101362‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Tumor necrosis factor receptors 1 and 2 are associated with early glomerular lesions in type 2 diabetes.

Elevated serum tumor necrosis factor receptor 1 (TNFR1) and 2 (TNFR2) concentrations are strongly associated with increased risk of end-stage renal disease in type 2 diabetes. However, little is known about the early glomerular structural lesions that develop in patients when these markers are elevated. Here we examined the relationships between TNFRs and glomerular structure in 83 American Indians with type 2 diabetes. Serum TNFRs and glomerular filtration rates (GFR, iothalamate) were measured during a research exam performed within a median of 0.9 months from a percutaneous kidney biopsy. Associations of TNFRs with glomerular structural variables were quantified by Spearman's correlations and by multivariable linear regression after adjustment for age, gender, diabetes duration, hemoglobin A1c, body mass index, and mean arterial pressure. The baseline mean age was 46 years, median GFR 130 ml/min, median albumin/creatinine ratio 26 mg/g, median TNFR1 1500 pg/ml, and median TNFR2 3284 pg/ml. After multivariable adjustment, TNFR1 and TNFR2 significantly correlated inversely with the percentage of endothelial cell fenestration and the total filtration surface per glomerulus. There were significant positive correlations with mesangial fractional volume glomerular basement membrane width, podocyte foot process width, and percent of global glomerular sclerosis. Thus, TNFRs may be involved in the pathogenesis of early glomerular lesions in diabetic nephropathy.

Podocyte Structural Parameters Do Not Predict Progression to Diabetic Nephropathy in Normoalbuminuric Type 1 Diabetic Patients

Background: Podocyte injury has been implicated in diabetic nephropathy (DN) ranging from normoalbuminuria to proteinuria in both type 1 and type 2 diabetes. Methods: To determine whether podocyte structural parameters predict DN risk in initially normoalbuminuric long-standing type 1 diabetic patients, we performed a nested case-control study in sex and diabetes duration-matched progressors (progression to proteinuria or ESRD, n = 10), non-progressors (normoalbuminuric at follow-up, n = 10), and non-diabetic controls (n = 10). Results: HbA_1c and diastolic blood pressure were higher in progressors versus non-progressors. Podocyte number per glomerulus, numerical density of podocyte per glomerulus, and foot process width were not different among groups. The glomerular basement membrane width was greater in progressors versus non-progressors or controls, and in non-progressors versus controls. As expected, the mesangial fractional volume was greater in progressors and non-progressors versus controls, with no differences between progressors and non-progressors. Conclusion: This study does not indicate that podocyte structural changes are preconditions for later DN progression in initially normoalbuminuric type 1 diabetic patients. However, this does not preclude an important role for podocyte injury at a later stage of DN.