Glomerular Basement Membrane Thickening Research Articles

#1656 Ezetimibe improves diabetic nephropathy by upregulating Nrf2 to alleviate endoplasmic reticulum stress

Abstract Background and Aims Diabetic nephropathy (DN), a prevalent complication of diabetes mellitus, stands as the primary contributor to chronic kidney disease and end-stage renal disease. The progression of diabetic nephropathy is facilitated by endoplasmic reticulum stress and the excessive generation of reactive oxygen species (ROS). In addition to its cholesterol-lowering properties, recent research has revealed that ezetimibe also ameliorates insulin resistance and glucose intolerance abnormalities, and is also beneficial in chronic kidney disease. However, the specific impact of ezetimibe on diabetic nephropathy and the underlying mechanism remain unknown. Therefore, the objective of this research is to investigate the effect of ezetimibe on diabetic nephropathy and elucidate its mechanism. Method In vivo experiments were conducted using C57BL/6J mice, with a total of 24 mice randomly assigned to three groups: NC, DM, and EZE (10 mg/kg/day). In vitro experiments involved the treatment of human renal proximal tubular (HK-2) cells with ezetimibe (50 μM) and high glucose (30 mmol/L) for 48 hours. Then, combined with histopathological examination, biochemical evaluation, TUNEL stain, western blot analysis, Si-RNA transfection, transmission electron microscopy and ROS probe were used to detect the renal morphology and function, oxidative stress and endoplasmic reticulum homeostasis level. Results The results demonstrated that a 10-week treatment with ezetimibe led to significant improvements in urine volume, beta-N-acetylglucosaminidase (NAG), and the urinary albumin/creatinine ratio (ACR). The administration of ezetimibe in the treatment exhibited enhancements in glomerular hypertrophy and renal tubular vacuolization, as well as mesangial matrix expansion and glomerular basement membrane thickness, in diabetic mice. Additionally, ezetimibe treatment resulted in a reduction in ROS formation by upregulating the expression of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), through the activation of NF-E2-related factor-2 (Nrf2). Furthermore, ezetimibe administration partially reversed the increased expression of endoplasmic reticulum stress-related proteins, including activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP), in DN mice. Ezetimibe demonstrated inhibitory effects on the accumulation of ROS and alleviated endoplasmic reticulum stress in HK-2 cells treated with high glucose. Furthermore, electron microscopy analysis revealed that ezetimibe effectively reduced the distension and dilation of the endoplasmic reticulum. However, the anti-endoplasmic reticulum stress effect of ezetimibe was significantly diminished following transfection with si-Nrf2. These findings suggest that ezetimibe exerts its anti-endoplasmic reticulum stress effect, at least in part, by enhancing antioxidant capacity and reducing ROS levels through the upregulation of Nrf2. Conclusion In summary, our research findings demonstrate the notable contribution of endoplasmic reticulum stress to the progression of DN. Through the up-regulation of Nrf2, ezetimibe effectively mitigates ROS generation in the context of DN, consequently ameliorating the adverse consequences of ERS, restoring equilibrium within the endoplasmic reticulum, and ultimately safeguarding against renal cell demise. Ezetimibe is an attractive potential therapy for the treatment of DN.

#234 Clinical spectrum and genetic variants of Alport syndrome and thin basement membrane nephropathy—single center experience

Abstract Background and aims Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetically heterogeneous kidney diseases that manifest with kidney involvement, AS also with hearing and visual impairment. Electron microscopic examination shows characteristic ultrastructural changes in the glomerular basement membrane (GBM) in AS and TBMN, but cannot reliably distinguish between the two entities. Therefore, molecular genetic studies and the identification of individual pathogenic variants in the target genes are crucial to confirm the diagnosis. The aim of our study is to determine the significance of the genetic variants associated with the ultrastructural changes of GBM found in the renal biopsy. Method We retrospectively analyzed records of patients from the University Medical Centre Ljubljana in which ultrastructural GBM changes characteristic for AS and TBMN were found in the renal biopsy, including their clinical, laboratory and histological features. Molecular genetic investigations were made using the new generation sequencing method (NGS) to determine germline pathogenic variants in genes directly related to AS and TBMN (COL4A3, COL4A4 and COL4A5) and in genes that are important in syndromes with a related clinical picture (COL4A6, CD151, LMX1B, MYH9, FN1 and MYO1E). The pathogenicity of variants was determined according to the Clinical Molecular Genetic Society best practice guidelines. Results The study included 73 patients with ultrastructurally confirmed thin GBM. Indications for kidney biopsy were chronic nephritis syndrome in 33 patients (45%), acute worsening of renal function in 16 (22%), nephrotic syndrome in 9 (12%), nephrotic proteinuria and chronic kidney disease in 6 (8%), isolated proteinuria in 5 (7%) and suspected vasculitis in 4 patients (6%). At the time of kidney biopsy average age was 50 ± 16 years, average creatinine 112 ± 70 µmol/L and average estimated glomerular filtration rate (eGFR) 65 ± 24 ml/min/1.73 m2, 7 patients had eGFR below 30 ml/min/1.73 m2. Average daily proteinuria was 2.4 ± 3.3 gr and 88% of patients had hematuria. 38% of them have hearing impairment and 43% had positive familiar history of kidney disease. We recorded 33 patients (45%) with genetic variants associated with AS or TBMN: 22 patients (67%) with pathogenic variants, 2 patients (6%) with likely pathogenic and 9 patients (27%) with variants of uncertain significance. The presence of a positive genetic analysis was associated with a higher level of hematuria (p = 0.003), a lower level of proteinuria (p = 0.004) and a positive family history of kidney disease (p < 0.001). It was also associated with hearing loss, but the association was not statistically significant (p = 0.099). Patients with genetic variants had similar eGFR to those with negative genetics (63.9 ml/min/1.73 m2 (58.3-69.4) vs 66.2 ml/min/1.73 m2 (60.0-72.3)), p = 0.698. We also found no differences in GBM thickness in patients without compared to those with identified gene variants (213 nm (160-315) vs. 209 nm (140-410)), p = 0.761. 32 patients (44%) had only focal chronic changes and ultrastructural changes of thin GBM present on kidney biopsy and 41 patients (56%) had also been diagnosed with other renal pathology. When comparing these two groups, patients without associated pathology had lower daily proteinuria (p = 0.048), higher level of hematuria (p = 0.021), frequent hearing impairment (p = 0.008) and also positive genetics analysis (p = 0.016). Conclusion We have shown that in patients with proven ultrastructural alterations in GBM, the presence of so far recognized genetic variants associated with AS and TBMN is 45%, and corresponds to the frequency of other phenotypic characteristics of AS, including hearing impairment and positive family history. In the future, additional clinical tools will be required to accurately determine the role of coexistent thin GBM on the prognosis of patients with other relevant kidney pathology and other genetic tools beyond NGS should be employed to explore other pathologic genetic variants associated with the AS spectrum.

A zebrafish model of diabetic nephropathy shows hyperglycemia, proteinuria and activation of the PI3K/Akt pathway.

Diabetic nephropathy (DN), as a complication of diabetes, is a substantial healthcare challenge owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement and glomerular sclerosis. Glomerular dysfunction was restored upon calorie restriction. RNA sequencing analysis demonstrated that DN zebrafish kidneys exhibited transcriptional patterns similar to those seen in human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating Akt phosphorylation. Our results indicate that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.

A compared histopathological study on kidneys and eye bulbs in distinct clinical presentations of canine leishmaniasis by Leishmania infantum.

Canine leishmaniasis (CanL) caused byLeishmania infantumcommonly progresses with renal and ophthalmic lesions associated with active systemic disease. As chronic inflammation related to immune complex deposits is a pathophysiological factor in the development of both glomerulonephritis and uveitis, we aimed to evaluate renal and ocular histopathological lesions and analyze whether they were related to each other and the clinical degree of the disease. For that, we evaluated 15 dogs from CanL-endemic areas.L. infantumPCR-positive dogs were studied according to disease severity into two different groups: Group-1 (G1) had data from seven dogs with mild to moderate CanL and no history of treatment, and G2 was formed with eight dogs with severe to terminal disease that had not responded to CanL treatment. Histopathological analysis of kidneys showed higher frequencies and intensities of glomerular basement membrane thickening (p = 0.026), deposits in glomeruli (p = 0.016), epithelial necrosis (p = 0.020), tubular dilatation (p = 0.003) and interstitial fibrosis (p = 0.04) in G2 dogs than in G1 dogs. Surprisingly, the histopathology of eye bulbs showed a higher frequency and intensity of retinitis (p = 0.019) in G1 dogs than in G2 dogs. The comparative analysis showed that there was no correspondence between histopathological findings in kidneys versus eyes in milder or more severe CanL. Our findings suggested that (1) clinically undetectable eye alterations can be more precocious than those in kidneys in the development of CanL, and (2) the lower frequency of eye lesions and higher frequency of renal lesions in dogs with terminal disease even after treatment indicate that therapy may have been effective in reducing CanL-associated ophthalmic disease but not proportionally in reducing kidney disease.

Unraveling a Novel Mechanism of Altered Glomerular Hemodynamics in Pre-diabetic Obesity

Background: In pre-diabetic obesity, disrupted glomerular autoregulation leads to increased glomerular capillary pressure and renal blood flow, causing potential barotrauma-induced glomerular damage. A decrease in Afferent arteriole ( Af-Art) resistance causes higher glomerular capillary pressure but the underlying mechanism behind this, specifically in pre-diabetic obesity, remains unclear. Hyperinsulinemia is common in pre-diabetic obesity, but its role in reducing Af-Art resistance in the kidneys is unknown. Kidney blood flow is auto-regulated by vasoconstriction via tubuloglomerular feedback (TGF) and vasodilation through the Distal convoluted tubule-2/connecting tubule (DCT-2/CNT) mechanism. Our earlier studies indicate increased Af-Art dilation in pre-diabetic obese rats through the DCT-2/CNT mechanism without affecting TGF. This DCT-2/CNT induced Af-Art dilation requires initiation from epithelial sodium channels (ENaC) present at this tubular segment and interestingly insulin is a known ENaC activator. Hypothesis: We hypothesize that in the context of pre-diabetic obesity, hyperinsulinemia contributes to an increase in renal cortical blood flow (CBF) by increasing DCT-2/CNT-induced Af-Art dilation, subsequently leading to glomerular injury. Methods: We assessed the effects of insulin on Af-Art dilation by perfusing juxtaposed DCT-2/CNT segments with insulin in normal rabbits. Contribution of DCT-2/CNT induced Af-Art dilation was measured by co-administration of the ENaC blocker benzamil. To study hyperinsulinemia's role in an in-vivo setting without altering blood glucose levels, we used a hyperinsulinemic-euglycemic (HI-EUG) clamp in obese rats with intravenous (IV) glucose and insulin infusions. Renal CBF and blood pressure were monitored using a laser Doppler probe and femoral artery catheter, respectively, both with and without benzamil administration (400 μg/kg, IV). To evaluate insulin's potential role in causation of renal damage, we infused insulin directly into one kidney of lean rats for 6 weeks via a renal sub-capsular catheter, with the contralateral kidney serving as control. Glomerular basement membrane (GBM) thickness, a marker of renal damage, was measured via electron microscopy. Untargeted lipidomic analysis of the renal cortex was performed to identify potential renal damage pathways in insulin infused kidneys. Results: Insulin perfusion in rabbits' DCT-2/CNT potentiated the dilation of adherent Af-Art compared to control infusions with artificial tubular fluid. ENaC inhibition reversed the insulin-induced Af-Art dilation, confirming that insulin increases Af-Art dilation through the ENaC-initiated DCT-2/CNT mechanism. In obese rats, insulin delivery using a HI-EUG clamp resulted in a significant 20.8±4.9% increase in CBF, which was negated by the administration of the ENaC blocker benzamil, indicating the potential involvement of DCT-2/CNT-mediated vasodilation. Neither the HI-EUG clamp nor benzamil infusion affected blood pressure. Additionally, continuous direct insulin infusion into the kidneys of lean rats led to an increase in glomerular basement membrane (GBM) thickness and ceramide accumulation in the renal cortex, both indicative of renal damage. Conclusion: Our study demonstrates that insulin directly augments DCT-2/CNT-mediated Af-Art dilation and increases renal cortical blood flow in obese rats. Chronic renal insulin infusion led to structural changes and damage markers in the kidney, independent of blood glucose and blood pressure levels. Henry Ford Health System Internal Funding. Pilot and Feasibility grant from southeast center for integrated metabolomics. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Correlation of light and electron microscopic morphometric parameters of glomerular capillaries with serum creatinine and proteinuria

ABSTRACT Waste products in the bloodstream are filtered by the glomerular capillaries in the kidneys and excreted into the urine. When making a differential diagnosis of kidney diseases, structural assessment of glomeruli using histological, ultrastructural, and immunological studies is crucial. This study assessed the microscopic and ultrastructural morphometric parameters of glomerular capillaries and examined their correlation with serum creatinine and proteinuria. A total of 60 kidney biopsy cases received by the transmission electron microscope (TEM) laboratory for diagnosis were included in the study. Toluidine blue stained 300 nm thick sections of TEM tissue blocks were scanned for glomerular morphometry by a whole slide imaging system, and the estimation of Bowman’s capsule (BC) area, glomerular capillary lumen diameter (GCLD), glomerular capillary density (GCD), glomerular capillary surface area density (GCSA), and percentage of glomerular capillary lumen space (%GCLS) was performed with QuPath software. TEM images of 70 nm thick sections were used for the evaluation of endothelial fenestration diameter (EFD), glomerular basement membrane (GBM) thickness, and podocyte foot process (PFP) effacement. Proteinuria and serum creatinine showed positive correlations with GBM thickness and PFP effacement. Negative correlations of serum creatinine were observed with EFD, %GCLS, and GCSA. Hence, glomerular filtration is greatly affected by the total area of the glomerular capillary surface and structural changes of GBM. Reduction of glomerulus filtration due to foot process effacement and thickening of GBM results in damage to the filtration barrier leading to the leakage of plasma protein into urine.

Amelioration of diabetic nephropathy in mice by a single intravenous injection of human mesenchymal stromal cells at early and later disease stages is associated with restoration of autophagy

Background and aimsMesenchymal stromal cells (MSCs) a potentially effective disease-modulating therapy for diabetic nephropathy (DN) but their clinical translation has been hampered by incomplete understanding of the optimal timing of administration and in vivo mechanisms of action. This study aimed to elucidate the reno-protective potency and associated mechanisms of single intravenous injections of human umbilical cord-derived MSCs (hUC-MSCs) following shorter and longer durations of diabetes.MethodsA streptozotocin (STZ)-induced model of diabetes and DN was established in C57BL/6 mice. In groups of diabetic animals, human (h)UC-MSCs or vehicle were injected intravenously at 8 or 16 weeks after STZ along with vehicle-injected non-diabetic animals. Diabetes-related kidney abnormalities was analyzed 2 weeks later by urine and serum biochemical assays, histology, transmission electron microscopy and immunohistochemistry. Serum concentrations of pro-inflammatory and pro-fibrotic cytokines were quantified by ELISA. The expression of autophagy-related proteins within the renal cortices was investigated by immunoblotting. Bio-distribution of hUC-MSCs in kidney and other organs was evaluated in diabetic mice by injection of fluorescent-labelled cells.ResultsCompared to non-diabetic controls, diabetic mice had increases in urine albumin creatinine ratio (uACR), mesangial matrix deposition, podocyte foot process effacement, glomerular basement membrane thickening and interstitial fibrosis as well as reduced podocyte numbers at both 10 and 18 weeks after STZ. Early (8 weeks) hUC-MSC injection was associated with reduced uACR and improvements in multiple glomerular and renal interstitial abnormalities as well as reduced serum IL-6, TNF-α, and TGF-β1 compared to vehicle-injected animals. Later (16 weeks) hUC-MSC injection also resulted in reduction of diabetes-associated renal abnormalities and serum TGF-β1 but not of serum IL-6 and TNF-α. At both time-points, the kidneys of vehicle-injected diabetic mice had higher ratio of p-mTOR to mTOR, increased abundance of p62, lower abundance of ULK1 and Atg12, and reduced ratio of LC3B to LC3A compared to non-diabetic animals, consistent with diabetes-associated suppression of autophagy. These changes were largely reversed in the kidneys of hUC-MSC-injected mice. In contrast, neither early nor later hUC-MSC injection had effects on blood glucose and body weight of diabetic animals. Small numbers of CM-Dil-labeled hUC-MSCs remained detectable in kidneys, lungs and liver of diabetic mice at 14 days after intravenous injection.ConclusionsSingle intravenous injections of hUC-MSCs ameliorated glomerular abnormalities and interstitial fibrosis in a mouse model of STZ-induced diabetes without affecting hyperglycemia, whether administered at relatively short or longer duration of diabetes. At both time-points, the reno-protective effects of hUC-MSCs were associated with reduced circulating TGF-β1 and restoration of intra-renal autophagy.Graphical abstract

Potential Renal Effects of Cigarette Smoking in the Diabetic State-A review

Diabetes is an alarming global systemic metabolic disorder that can pose a major threat to patients. The serious consequences of cigarette smoking on the diabetic kidney are not well known among people in different countries. According to different studies, smoking enhances albuminuria in diabetic patients. On the other hand, urinary albumin is a sensitive indicator of glomerular injury. The abnormal trans-glomerular passage of albumin may be seen due to increased permeability of the glomerular capillary wall and their subsequent impaired reabsorption by the epithelial cells of the proximal tubule. Smoking with hyperglycemia increases lipid accumulation and oxidative stress, which mainly up-regulates TGF-β, accumulates AGEs, reduces nitric oxide production, and eventually causes glomerular basement membrane thickening and mesangial expansion that results in the development of glomerulosclerosis and nephropathy. The complex interaction between cigarette smoking and diabetic mellitus poses multiple challenges for researchers, physicians, and patients. Therefore, the present review article aims to find out the feasible consequences for the kidney of a diabetic patient due to the habit of cigarette smoking which may be useful for academicians and researchers in the future.

Chrysophanol ameliorates oxidative stress and pyroptosis in mice with diabetic nephropathy through the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 signaling pathway.

Diabetic nephropathy (DN), a microvascular complication of diabetes, increases the risk of all-cause diabetes and cardiovascular mortalities. Moreover, oxidative stress and pyroptosis play important roles in the pathogenesis of DN. Rhubarb is widely used in traditional medicine, and chrysophanol (Chr), a free anthraquinone compound abundant in rhubarb, exhibits potent antioxidant properties and ameliorates renal fibrosis. Therefore, this study aimed to investigate the effects of Chr on renal injury, oxidative stress, and pyroptosis in mice with DN. A DN model was established by feeding the mice a high-sugar and fat diet and injecting them with 50 mg/kg streptozotocin as a positive control. The DN mice had significantly impaired renal function, thickened glomerular thylakoids and basement membranes, increased fibrous tissue, and inflammatory cell infiltration. Superoxide dismutase (SOD) levels were reduced, malondialdehyde (MDA) levels were increased, interleukin (IL)-1β and IL-18 increased, and cleaved caspase-1, caspase-1, and gasdermin D (GSDMD) involved in the process of pyroptosis were upregulated in DN. Kelch-like ECH-associated protein 1 (Keap1) expression was upregulated, and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was downregulated. Compared to those in the DN group, the Chr-treated mice with DN had improved renal dysfunction, weakened glomerular thylakoid and basement membrane thickening, and reduced fibrous tissue proliferation and inflammatory cell infiltration. Additionally, Chr increased SOD levels, decreased MDA, IL-1β, and IL-18, down-regulated caspase-1, cleaved caspase-1, GSDMD, and Keap1 expression, and upregulated Nrf2 expression, which reversed the DN. Therefore, Chr reduced oxidative stress and pyroptosis in DNmice by activating the Keap1/Nrf2 pathway.

Chia seeds and coenzyme Q10 alleviate iron overload induced hepatorenal toxicity in mice via iron chelation and oxidative stress modulation

Iron overload (IOL) can cause hepatorenal damage due to iron-mediated oxidative and mitochondrial damage. Remarkably, combining a natural iron chelator with an antioxidant can exert greater efficacy than monotherapy. Thus, the present study aimed to evaluate the efficacy of Chia and CoQ10 to chelate excess iron and prevent hepatorenal oxidative damage in IOL mice. Male Swiss albino mice (n = 49) were randomly assigned to seven groups: control, dietary Chia, CoQ10, IOL, IOL + Chia, IOL + CoQ10, and IOL + Chia + CoQ10. Computational chemistry indicates that the phytic acid found in the Chia seeds is stable, reactive, and able to bind to up to three iron ions (both Fe2+ and Fe3+). IOL induced a significant (P < 0.05) increase in serum iron, ferritin, transferrin, TIBC, TSI, RBCs, Hb, MCV, MCH, WBCs, AST, ALT, creatinine, and MDA. IOL causes a significant (P < 0.05) decrease in UIBC, platelets, and antioxidant molecules (GSH, SOD, CAT, and GR). Also, IOL elicits mitochondrial membrane change depolarization, and DNA fragmentation and suppresses mitochondrial DNA copies. Furthermore, substantial changes in hepatic and renal tissue, including hepatocellular necrosis and apoptosis, glomerular degeneration, glomerular basement membrane thickening, and tubular degeneration, were observed in the IOL group. Dietary Chia and CoQ10 induced significant (P < 0.05) amelioration in all the mentioned parameters. They can mostly repair the abnormal architecture of hepatic and renal tissues induced by IOL, as signified by normal sinusoids, normal central veins, and neither glomerular damage nor degenerated tubules. In conclusion, the combined treatment with Chia + CoQ10 exerts more pronounced efficacy than monotherapy in hepatorenal protection via chelating excess iron and improved cellular antioxidant status and hepatorenal mitochondrial function in IOL mice.

Both sexes develop DKD in the CD1 uninephrectomized streptozotocin mouse model

Diabetic kidney disease (DKD) is characterized by a progressive increase in albuminuria and typical pathologic features. Recent studies have shown that sex is an important factor to consider in the pathogenesis of DKD. Presently, the hallmarks of this disease have primarily been studied in male rodent models. Here we explored the influence of sex in a murine model of DKD. CD1 mice underwent a right nephrectomy followed by intraperitoneal injection with 200 mg/kg streptozotocin to induce type 1 diabetes. Due to a high mortality rate, females required a reduction in streptozotocin to 150 mg/kg. Mice were followed for 12 weeks. Both sexes developed comparable hyperglycemia, while albuminuria and glomerular volume were increased to a greater degree in females and kidney hypertrophy was only seen in females. Males had a greater increase in blood pressure and glomerular basement membrane thickening, and a greater decrease in endpoint weight. Serum TGFβ1 levels were increased only in females. However, both sexes showed a similar increase in induction of kidney fibrosis. T cell and macrophage infiltration were also increased in both sexes. While some differences were observed, overall, both sexes developed clinical and pathologic characteristics of early DKD. Future studies evaluating therapeutic interventions can thus be assessed in both sexes of this DKD model.

Renal biopsy pattern in diabetes mellitus patients and their correlation with clinical parameters

Background. Diabetic nephropathy is a condition marked by persistent proteinuria, hypertension, and a progressive loss of renal function. End-stage kidney disease needing continuous renal replacement treatment is now primarily caused by diabetes. According to Kimmelstiel and Wilson, the hallmark lesion of diabetic nephropathy is nodular glomerulosclerosis. Diabetic nephropathy or Nondiabetic renal disease, or the coexistence of both can be seen in renal histopathology and in differentiating between these diagnostic groups can have an impact on patient care and prognosis.Patients and Methods. Total of 21 cases of Diabetic nephropathy were included in the study. Clinical details and laboratory parameters like diastolic blood pressure, creatinine level, 24 hrs urinary protein level and HbA1C% were recorded in pretested performa in all cases. The biopsy specimens were stained with hematoxylin &amp; eosin and special stains.Results. Among the total DM cases only 21 patients have done renal biopsy, 11 cases (52.3 %) showed KW lesion (Class III) while 06 cases (28.5 %) showed diffuse diabetic glomerulosclerosis (Class IV). The remaining 04 cases (19 %) showed a mild increase in mesangial matrix and slight thickening of glomerular basement membrane (Class II). When compared with clinical parameters, they were more raised in Nodular diabetic glomerulosclerosis type (Class III) lesion as compared to diffuse diabetic glomerulosclerosis.Conclusion. Nodular diabetic glomerulosclerosis was the most common lesion in renal biopsy of type II diabetes mellitus patients. This KW lesion is responsible for more severe clinical and biochemical renal abnormality in most patients with type II diabetes mellitus.

Study of Neutrophil–Lymphocyte Ratio, Interleukin-6 (IL-6) and Tumor Necrosis Factor-Alpha (TNF-α) in Type 2 Diabetes with Diabetic Nephropathy

Abstract Background Diabetic kidney disease is defined by characteristic structural and functional changes. The predominant structural changes include mesangial expansion, glomerular basement membrane thickening, podocyte injury, and, ultimately, glomerular sclerosis. The major clinical manifestations of diabetic nephropathy are albuminuria, hematuria (less often), and, in many patients, progressive chronic kidney disease, which can be slowed with optimal therapy. Objective to assess the Neutrophil-lymphocyte Ratio, Interleukin- 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in Type 2 Diabetes with Diabetic Nephropathy. Patients and Methods In Our study We collect 90 subjects with their ages ranging from 35-70, selected from Internal Medicine and Endocrinology outpatient clinics and the inpatient department at Ain Shams University Hospitals. Results As regards NLR, there were statistically significant and direct correlations between NLR and IL6, TNF, serum BUN, serum creatinine and albumin to creatinine ratio, while significant and indirect correlation with EGFR. As regards IL6, there were statistically significant and direct correlations between IL6 and TNF, serum BUN, serum creatinine and albumin to creatinine ratio, while significant and indirect correlation with BMI and EGFR. As regards TNF, there were statistically significant and direct correlations between TNF and serum BUN and albumin to creatinine ratio. There is no statistical difference between the studied groups as regard sex, age, BMI, SBP, DBP. Conclusion The current study could find that NLR, IL-6 and TNF-α were significantly associated with albuminuria in Type 2 Diabetic patients with DN and that they can be used as evident biomarkers in assessing the progression of deteriorating renal function and different stages of renal disease in patients with DN.

Deletion of protein tyrosine phosphatase SHP-1 restores SUMOylation of podocin and reverses the progression of diabetic kidney disease

Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the invivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-β, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.

The kidney antifibrotic effects of 5,7,3′,4′,5′-pentamethoxyflavone from Bauhinia championii in streptozotocin-induced diabetic rats: in vivo and in vitro experiments

Context The antidiabetic effects of flavonoids have been reported, but it is still unclear whether 5,7,3′,4′,5′-pentamethoxyflavone, isolated from Bauhinia championii Benth. (Fabaceae), also exhibits such properties. Objective To isolate 5,7,3′,4′,5′-pentamethoxyflavone from B. championii using high-speed countercurrent chromatography and examine its potential in treating diabetic nephropathy. Materials and methods The phytochemical constituents from the stems of B. championii were separated and purified with high-speed countercurrent chromatography; 5,7,3′,4′,5′-pentamethoxyflavone (PMF) was identified by mass spectrum, 1H-NMR, and 13C-NMR. After exposing mesangial cells to 30 mM glucose and either 5 μM or 10 μM PMF for 6 h, the levels of fibronectin (FN) and p-Smad2/3 were analyzed using Western blotting. Male Sprague–Dawley rats were injected intraperitoneally with 55 mg/kg streptozotocin to induce diabetes and then were randomized into three groups (n = 10): vehicle administration, low-dose (5 mg/kg) PMF, and high-dose (25 mg/kg) PMF by intragastric gavage for 3 months. A healthy group was included as the control. Results Compared to the diabetic group, low-dose and high-dose PMF treatment decreased the phosphorylation of Smad2/3 by 0.54- and 0.52-fold, and the accumulation of FN decreased by 0.82- and 0.77-fold in vitro; the phosphorylation of Smad2/3 was decreased by 0.39- and 0.37-fold, and the accumulation of FN decreased by 0.47- and 0.40-fold in vivo, respectively. Furthermore, PMF alleviated the glomerular basement membrane thickness and foot process fusion. Conclusion The findings suggest for the first time that PMF may be a promising treatment option for diabetic kidney fibrosis, which warrants additional clinical investigation.

401-P: Effects of Teneligliptin on Lipotoxicity in High-Fat Diet–Induced Diabetic Kidney Disease Model

Lipotoxicity contributes to diabetic kidney disease (DKD), impairing function of glomerulus and podocyte, consequently causing albuminuria. Dipeptidyl peptidase-4 (DPP-4) inhibitors demonstrated renoprotective effects in DKD, but the mechanism for protection in DKD regarding lipid metabolism is unclear. Here, we investigated the effects of teneligliptin in high-fat diet (HFD)-induced DKD model. 5-week-old male C57BL/6J mice were supplemented with HFD for 24 weeks to develop DKD. Teneligliptin was administrated orally by pre-mixing with HFD for 16 weeks. Body weight, blood glucose levels, and glucose and insulin tolerance test were evaluated at 12 weeks and albuminuria was evaluated at 6 and 12 weeks after teneligliptin administration. Lipid panels were evaluated and the renal cortex was collected for histologic examination at 16 weeks. Body weight, random and fasting blood glucose was not significantly different but total cholesterol and triglyceride were reduced in teneligliptin group. Glucose and insulin tolerance test showed significant improvement in teneligliptin group compared with HFD group (p&amp;lt;0.001 and p=0.009, respectively). Changes in albuminuria between 6 and 12 weeks during treatment period group were lower in teneligliptin group compared with HFD group (p=0.07). In immunohistochemistry studies, glomerular volume and mesangial expansion were attenuated in the teneligliptin group by 26.9% and 32.15% respectively (p=0.011 and p=0.043, respectively). Glomerular basement membrane thickness and foot process width were decreased by 43.2% and 9.32% in teneligliptin group (p=0.0007 and p=0.035, respectively). Teneligliptin attenuated diabetic kidney disease in HFD-induced DKD by preserving integrity of glomerulus and podocyte, with decreased albuminuria. Further investigations for detailed mechanisms about the association between DPP-4 inhibitor and lipotoxicity induced glomerulus and podocyte injury in DKD are warranted in the future. Disclosure H.Kim: None. E.Kang: None. R.Kim: None. Y.Yang: None. H.Park: None. N.Jeon: None. M.Lee: None. H.Lee: None. B.Lim: None. H.Choi: None.

#4488 OLD BIOPSY IS ALWAYS GOLD, REVISING MEMBRANOPROLIFERATIVE GN DIAGNOSIS IN A RENAL TRANSPLANT PATIENT

Abstract Background and Aims MPGN resulting from hepatitis C virus (HCV) infection typically shows granular deposition of immunoglobulin M (IgM), C3, and both kappa and lambda light chains. Immunoglobulin G (IgG) may or may not be present, and C1q is typically negative, the same pathology can be found in other viral infections. The recurrence rate post-transplantation is high, with 33% of immune-complex MPGN. Graft loss is even higher 65%, especially among patients with idiopathic MPGN. Treatment of a patient with significant proteinuria can be tricky, as no proven treatment is described yet, but plasmapheresis and induction with cyclophosphamide were described for heavy proteinuria &amp;gt;3.5 g/day. Method 79 year old female patient, known case of live unrelated renal transplantation in December 2013, primary renal disease is unknown. She had a history of equivocal hepatitis C virus antibodies, but Hepatitis C virus RNA was not detected. She is also known to have Type II Diabetes Mellitus and hypertension. The patient had baseline creatinine of 100-120 micromol/L. 24 hr urine protein was 0.8 g/Day but started rising quickly to reach a peak of 9.2 g/g seven months post-transplantation to nephrotic range proteinuria but her serum albumin level was always above 35 g/L. Serum protein electrophoresis showed no M band, a kappa/ Lambda ratio 1.21 within a normal range (0.260-1.65). Anti- nuclear antibody (ANA), P-ANCA, C-ANCA, and Double-stranded DNA all were negative. Complements were within normal range C3 1.39 g/L (0.79 -1.52), C4 0.44 g/L(0.17-0.57)m rheumatoid factor and Anti- citrullinated peptide antibodies were negative. Hepatitis b Virus surface antigen and core antibodies were not reactive, HIV screening was negative. Urine analysis showed RBCs but no RBC casts. Renal biopsy showed mild mesangial expansion and mild l increase in mesangial cells and matrix. Some glomeruli show increased lobulation with increase in mesangial cells and matrix with segmental endocapillary proliferation There is double contour of the glomerular basement membrane with scattered spikes and holes within the basement membrane seen best with silver stain. There is mild increase in the thickness of glomerular basement membrane. There is focal mild lymphocytic infiltration. Tubulitis is not seen in the examined material. One Focal area of mild interstitial fibrosis. Sampled blood vessels are unremarkable. The stain for C4d is negative. Immunofluorescence showed granular capillary wall immunoreactivity to IgM (+2), C4 (+1), C1q (+1), and Lambda light chain (+2). The glomeruli show no immunoreactivity to IgG, IgA, C3, Kappa and fibrin. The glomeruli show (+) staining for albumin. Tubular protein reabsorption droplets showed immunoreactivity for albumin, IgG, IgM, C3, kappa and lambda light chains. Electron microscopy showed many subendothelial, and mesangial electron-dense deposits. The patient was on maintenance immunosuppression medications inform of mycophenolic acid 720 mg, cyclosporine; 250mg BID, and Prednisolone 5 mg daily.Revisiting the new classification of membranoproliferative glomerulonephritis is challenging in this patient, since there is the dominance of IgM deposition by immunofluorescence and C4 but not C3 makes it challenging, the negative monoclonal band in serum electrophoresis and negative autoimmune workup excludes immune complex mediate MPGN, leave idiopathic MPGN a possibility, keeping in mind that hepatitis C virus infection was never confirmed. On the other hand, IF showed IgM with C4 subendothelial and mesangial dense deposits that excludes away the C4 glomerulopathy. Results Whether this is a recurrence of glomerular disease early on post-transplantation or denovo membranoproliferative disease, she responded well to the addition of losartan 100 mg daily, with a reduction of her urinary protein to less than 1 g/g. the patient never received treatment for the hepatitis C virus. Conclusion In our patient, it is difficult to conclude if the cause of MPGN is recurrence or denovo GN, especially with severe proteinuria that responded well to ACEi treatment. The pathological findings can be tricky.

Effects and mechanisms of total flavones of Abelmoschus manihot in improving insulin resistance and podocyte epithelial-mesenchymal transition in diabetic kidney disease based on IRS1/PI3K/Akt pathway

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.

Functional and morphological renal changes in a Göttingen Minipig model of obesity-related and diabetic nephropathy

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6–7 months (T1) and 12–13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.

Urokinase Plasminogen Activator Deficiency Aggravates Cationic Bovine Serum Albumin–Induced Membranous Nephropathy Through T Helper Cell Type 2-Prone Immune Response in Mice

Urokinase plasminogen activator (uPA) is a crucial activator of the fibrinolytic system that modulates tissue remodeling, cancer progression, and inflammation. However, its role in membranous nephropathy (MN) remains unclear. To clarify this issue, an established BALB/c mouse model mimicking human MN induced by cationic bovine serum albumin (cBSA), with a T helper cell type 2–prone genetic background, was used. To induce MN, cBSA was injected into Plau knockout (Plau−/−) and wild-type (WT) mice. The blood and urine samples were collected to measure biochemical parameters, such as serum concentrations of immunoglobulin (Ig)G1 and IgG2a, using enzyme-linked immunoassay. The kidneys were histologically examined for the presence of glomerular polyanions, reactive oxygen species (ROS), and apoptosis, and transmission electron microscopy was used to examine subepithelial deposits. Lymphocyte subsets were determined using flow cytometry. Four weeks post–cBSA administration, Plau−/− mice exhibited a significantly higher urine protein-to-creatine ratio, hypoalbuminemia, and hypercholesterolemia than WT mice. Histologically, compared to WT mice, Plau−/− mice showed more severe glomerular basement thickening, mesangial expansion, IgG granular deposition, intensified podocyte effacement, irregular thickening of glomerular basement membrane and subepithelial deposits, and abolishment of the glycocalyx. Moreover, increased renal ROS levels and apoptosis were observed in Plau−/− mice with MN. B-lymphocyte subsets and the IgG1-to-IgG2a ratio were significantly higher in Plau−/− mice after MN induction. Thus, uPA deficiency induces a T helper cell type 2–dominant immune response, leading to increased subepithelial deposits, ROS levels, and apoptosis in the kidneys, subsequently exacerbating MN progression in mice. This study provides a novel insight into the role of uPA in MN progression.