Early-Onset albuminuria and Associated Renal Pathology in Leucine-Rich Repeat Kinase 2 Knockout Rats.

Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

Empagliflozin protects glomerular endothelial cell architecture in experimental diabetes through the VEGF-A/caveolin-1/PV-1 signaling pathway.

In addition to having blood glucose-lowering effects, inhibitors of sodium glucose cotransporter 2 (SGLT2) afford renoprotection in diabetes. We sought to investigate which components of the glomerular filtration barrier could be involved in the antiproteinuric and renoprotective effects of SGLT2 inhibition in diabetes. BTBR (black and tan, brachyuric) ob/ob mice that develop a type 2 diabetic nephropathy received a standard diet with or without empagliflozin for 10 weeks, starting at 8 weeks of age, when animals had developed albuminuria. Empagliflozin caused marked decreases in blood glucose levels and albuminuria but did not correct glomerular hyperfiltration. The protective effect of empagliflozin against albuminuria was not due to a reduction in podocyte damage as empagliflozin did not affect the larger podocyte filtration slit pore size nor the defective expression of nephrin and nestin. Empagliflozin did not reduce the thickening of the glomerular basement membrane. In BTBR ob/ob mice, the most profound abnormality seen using electron microscopy was in the endothelial aspect of the glomerular capillary, with significant loss of endothelial fenestrations. Remarkably, empagliflozin ameliorated the subverted microvascular endothelial ultrastructure. Caveolae and bridging diaphragms between adjacent endothelial fenestrae were seen in diabetic mice and associated with increased expression of caveolin-1 and the appearance of PV-1. These endothelial abnormalities were limited by the SGLT2 inhibitor. Although no expression of SGLT2 was found in glomerular endothelial cells, SGLT2 was expressed in the podocytes of diabetic mice. VEGF-A, which is a known stimulus for endothelial caveolin-1 and PV-1, was increased in podocytes of BTBR ob/ob mice and normalized by SGLT2 inhibitor treatment. Thus, empagliflozin's protective effect on the glomerular endothelium of diabetic mice could be due to a limitation of the paracrine signaling of podocyte-derived VEGF-A that resulted in a reduction of the abnormal endothelial caveolin-1 and PV-1, with the consequent preservation of glomerular endothelial function and permeability. © 2022 The Pathological Society of Great Britain and Ireland.

Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine.

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

[Non-Classical Clinical Types and Pathological Changes of Diabetic Kidney Disease: A Review]. / ç³–å°¿ç— è‚¾è„ç–¾ç— éžç»å ¸ä¸´åºŠç±»åž‹ä¸Žç— ç†å˜åŒ–ç»¼è¿°.

Diabetic kidney disease (DKD) is a common complication of diabetes mellitus and approximately 1/3 of diabetic patients may progress to DKD. A typical early clinical manifestation of DKD is microalbuminuria and patients may present with macroproteinuria accompanied by a decrease in renal function condition as the disease progresses. It is generally believed that the likelihood of a reversal of the disease is reduced after the development of macroproteinuria in patients with DKD, and that eventually some patients' condition may develop into end-stage renal disease (ESRD). Moreover, the thickening of the glomerular basement membrane, mesangial matrix expansion, Kimmelstiel-Wilson (K-W) nodules, and glomerulosclerosis in end-stage diabetes mellitus are typical pathologic changes of DKD. However, some DKD patients, especially those with type 2 diabetes mellitus (T2DM) combined with DKD, may have diverse clinical manifestations, showing variations in disease progression and regression, and manifesting as non-classical types of DKD, such as normoalbuminuric DKD, proteinuria-reduced DKD, and DKD with rapid decline in renal function. In addition, the formation of crescents, a special pathological change, is observed in renal biopsy. However, this issue is currently under-recognized by clinicians and therefore deserves more attention. In order to improve clinicians' understanding of the presentations and pathological changes of non-classical DKD and the level of DKD prevention and treatment in China, we present a preliminary introduction to the clinical phenotypes and pathological changes of non-classical types of DKD in this paper by summarizing the findings of our prior studies as well as domestic and international literature.

Transient receptor potential cation channel 6 contributes to kidney injury induced by diabetes and hypertension.

Diabetes mellitus (DM) and hypertension (HTN) are major risk factors for chronic kidney injury, together accounting for >70% of end-stage renal disease. In this study, we assessed whether DM and HTN interact synergistically to promote kidney dysfunction and whether transient receptor potential cation channel 6 (TRPC6) contributes to this synergism. In wild-type (WT; B6/129s background) and TRPC6 knockout (KO) mice, DM was induced by streptozotocin injection to increase fasting glucose levels to 250-350 mg/dL. HTN was induced by aorta constriction (AC) between the renal arteries. AC increased blood pressure (BP) by ∼25 mmHg in the right kidney (above AC), whereas BP in the left kidney (below AC) returned to near normal after 8 wk, with both kidneys exposed to the same levels of blood glucose, circulating hormones, and neural influences. Kidneys of WT mice exposed to DM or HTN alone had only mild glomerular injury and urinary albumin excretion. In contrast, WT kidneys exposed to DM plus HTN (WT-DM + AC mice) for 8 wk had much greater increases in albumin excretion and histological injury. Marked increased apoptosis was also observed in the right kidneys of WT-DM + AC mice. In contrast, in TRPC6 KO mice with DM + AC, right kidneys exposed to the same levels of high BP and high glucose had lower albumin excretion and less glomerular damage and apoptotic cell injury compared with right kidneys of WT-DM + AC mice. Our results suggest that TRPC6 may contribute to the interaction of DM and HTN to promote kidney dysfunction and apoptotic cell injury.NEW & NOTEWORTHY A major new finding of this study is that the combination of moderate diabetes and hypertension promoted marked renal dysfunction, albuminuria, and apoptotic cell injury, and that these effects were greatly ameliorated by transient receptor potential cation channel 6 deficiency. These results suggest that transient receptor potential cation channel 6 may play an important role in contributing to the interaction of diabetes and hypertension to promote kidney injury.

The ability of remaining glomerular podocytes to adapt to the loss of their neighbours decreases with age.

Progressive podocyte loss is a feature of healthy ageing. While previous studies have reported age-related changes in podocyte number, density and size and associations with proteinuria and glomerulosclerosis, few studies have examined how the response of remaining podocytes to podocyte depletion changes with age. Mild podocyte depletion was induced in PodCreiDTR mice aged 1, 6, 12 and 18 months via intraperitoneal administration of diphtheria toxin. Control mice received intraperitoneal vehicle. Podometrics, proteinuria and glomerular pathology were assessed, together with podocyte expression of p-rp-S6, a phosphorylation target that represents activity of the mammalian target of rapamycin (mTOR). Podocyte number per glomerulus did not change in control mice in the 18-month time period examined. However, control mice at 18 months had the largest podocytes and the lowest podocyte density. Podocyte depletion at 1, 6 and 12 months resulted in mild albuminuria but no glomerulosclerosis, whereas similar levels of podocyte depletion at 18 months resulted in both albuminuria and glomerulosclerosis. Following podocyte depletion at 6 and 12 months, the number of p-rp-S6 positive podocytes increased significantly, and this was associated with an adaptive increase in podocyte volume. However, at 18 months of age, remaining podocytes were unable to further elevate mTOR expression or undergo hypertrophic adaptation in response to mild podocyte depletion, resulting in marked glomerular pathology. These findings demonstrate the importance of mTORC1-mediated podocyte hypertrophy in both physiological (ageing) and adaptive settings, highlighting a functional limit to podocyte hypertrophy reached under physiological conditions.

Renal deposition and clearance of recombinant poly-IgA complexes in a model of IgA nephropathy.

IgA nephropathy (IgAN) is the most common type of glomerulonephritis worldwide, which follows a chronic but nonetheless highly variable course of progression. IgA immune complexes are the primary source of renal deposits in IgAN. Apart from the presence of granular IgA1 deposits in the glomerular mesangium and mesangial hypercellularity as common features, the detailed process of IgA1 deposition and clearance in the kidney remains unclear. We sought to examine the dynamics of IgA deposition and tissue plasticity in response to deposits including their intrarenal clearance. We followed a synthetic approach to produce a recombinant fusion between IgA Fc (rIgA) and a biotin tag, which was subsequently induced with streptavidin (SA) to form an oligomeric poly-IgA mimic. Both uninduced rIgA (mono-rIgA) and polymeric SA-rIgA (poly-rIgA) were injected intravenously into Wistar rats. Plasma IgA levels and renal and liver histology were examined in a time series. In contrast to mono-rIgA, this synthetic poly-rIgA analog formed renal deposits exclusively in the glomerulus and were mostly cleared in 3 h. However, repeated daily injections for 12 days caused long-lasting and stronger glomerular IgA deposition together with IgG and complement C3, in association with mesangial cell proliferation, matrix expansion, and variable degrees of albuminuria and hematuria that phenocopied IgAN. Ex vivo, poly-rIgA bound cultured mesangial cells and elicited cytokine production, in addition to activating plasma C3 that was consistent with the actions of IgA immune complexes in IgAN pathogenesis. Remarkably, the kidneys were able to reverse all pathologic manifestations and restore normal glomerular histology 2 weeks after injections were halted. The synthetic model showed the kinetics between the intricate balance of renal deposition and clearance, as well as glomerular plasticity towards healing. Together, the results revealed a priming effect of existing deposits in promoting stronger and longer-lasting IgA deposition to cause renal damage. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Diabetic Kidney Disease: From Pathogenesis to Novel Treatment Possibilities.

One of the microvascular complications of diabetes is diabetic kidney disease (DKD), often leading to end stage renal disease (ESRD) in which patients require costly dialysis or transplantation. The silent onset and irreversible progression of DKD are characterized by a steady decline of the estimated glomerular filtration rate, with or without concomitant albuminuria. The diabetic milieu allows the complex pathophysiology of DKD to enter a vicious cycle by inducing the synthesis of excessive amounts of reactive oxygen species (ROS) causing oxidative stress, inflammation, and fibrosis. As no cure is available, intensive research is required to develop novel treatments possibilities. This chapter provides an overview of the important pathomechanisms identified in diabetic kidney disease, the currently established therapies, as well as recently developed novel therapeutic strategies in DKD.

Discovery of endoplasmic reticulum calcium stabilizers to rescue ER-stressed podocytes in nephrotic syndrome.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Activation of G protein-coupled estrogen receptor 1 ameliorates proximal tubular injury and proteinuria in Dahl salt-sensitive female rats.

Recent evidence indicates a crucial role for G protein-coupled estrogen receptor 1 (GPER1) in the maintenance of cardiovascular and kidney health in females. The current study tested whether GPER1 activation ameliorates hypertension and kidney damage in female Dahl salt-sensitive (SS) rats fed a high-salt (HS) diet. Adult female rats were implanted with telemetry transmitters for monitoring blood pressure and osmotic minipumps releasing G1 (selective GPER1 agonist, 400 µg/kg/day ip) or vehicle. Two weeks after pump implantation, rats were shifted from a normal-salt (NS) diet (0.4% NaCl) to a matched HS diet (4.0% NaCl) for 2 wk. Twenty-four hour urine samples were collected during both diet periods and urinary markers of kidney injury were assessed. Histological assessment of kidney injury was conducted after the 2-wk HS diet period. Compared with values during the NS diet, 24-h mean arterial pressure markedly increased in response to HS, reaching similar values in vehicle-treated and G1-treated rats. HS also significantly increased urinary excretion of protein, albumin, nephrin (podocyte damage marker), and KIM-1 (proximal tubule injury marker) in vehicle-treated rats. Importantly, G1 treatment prevented the HS-induced proteinuria, albuminuria, and increase in KIM-1 excretion but not nephrinuria. Histological analysis revealed that HS-induced glomerular damage did not differ between groups. However, G1 treatment preserved proximal tubule brush-border integrity in HS-fed rats. Collectively, our data suggest that GPER1 activation protects against HS-induced proteinuria and albuminuria in female Dahl SS rats by preserving proximal tubule brush-border integrity in a blood pressure-independent manner.

Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease.

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER-associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD-related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)-GFP knock-in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD.

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress.

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.

Enteropeptidase inhibitor SCO-792 effectively prevents kidney function decline and fibrosis in a rat model of chronic kidney disease.

BACKGROUND: Inhibiting enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of enteropeptidase inhibition on kidney parameters are largely unknown. Here, we evaluated the chronic effects of an enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model. METHODS: SCO-792, an orally available enteropeptidase inhibitor, was administered [0.03% and 0.06% (w/w) in the diet] to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR) for five weeks. The effects of SCO-792 and the contribution of amino acids to these effects were evaluated. RESULTS: SCO-792 increased the faecal protein content, indicating that SCO-792 inhibited enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria and renal histological damage than SCO-792 treatment, indicating the enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria. CONCLUSIONS: SCO-792-mediated inhibition of enteropeptidase potently prevented GFR decline, albuminuria and kidney fibrosis; hence, it may have therapeutic potential against CKD.

Deterioration of diabetic nephropathy via stimulating secretion of cytokines by atrial natriuretic peptide.

BACKGROUND: Atrial natriuretic peptide (ANP) is a cardiovascular and metabolic hormone that has been identified recently as being associated with chronic kidney disease (CKD) without diabetes. Cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and adiponectin (ADP) contribute to the development of type 2 diabetes (T2DM). The aim here was to investigate the relationships of ANP with cytokine levels and clinical variables in T2DM nephropathy patients. METHODS: A total of 81 participants with T2DM were recruited, including 37 patients with normoalbuminuria, 23 patients with microalbuminuria and 21 patients with macroalbuminuria. Serum concentrations of ANP and cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. The correlations between ANP and clinical variables were analyzed. Multiple linear regression and logistic regression models were constructed to test the associations between ANP and the severity and presence of albuminuria. RESULTS: The macroalbuminuria patients exhibited higher plasma levels of ANP, TNF-α, IL-6, and ADP; higher serum creatinine (Cr) and blood urea nitrogen (BUN); and longer duration of diabetes mellitus (DM) than the patients with normoalbuminuria and microalbuminuria. Plasma ANP level was significantly associated with TNF-α (r = 0.876, p < 0.001), IL-6 (r = 0.816, p < 0.001) and ADP (r = 0.772, p < 0.001), independent of the duration of DM or the BUN concentration. CONCLUSION: ANP is higher in type 2 diabetes mellitus nephropathy subjects, especially those who have macroalbuminuria, which is associated with compensatory responses to inflammation.

Non-invasive fibrosis assessments of non-alcoholic fatty liver disease associated with low estimated glomerular filtration rate among CKD patients: the Fukuoka Kidney disease Registry Study.

BACKGROUND: A growing body of evidence has shown that non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). Non-invasive fibrosis assessments of NAFLD such as Fibrosis-4 (FIB-4) index and NAFLD fibrosis score (NFS) have been developed to substitute liver biopsy. Little is known about the association between FIB-4 index or NFS and the components of CKD. METHODS: In the present cross-sectional study, we assessed of 3640 Japanese CKD patients. We examined the association between FIB-4index or NFS and the odds of having low estimated glomerular filtration rate (eGFR) defined as eGFR < 60 mL/min/1.73 m2 or albuminuria defined as urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g. Patients were divided into quartiles according to their baseline FIB-4 index and NFS levels. Linear and logistic regression analysis were conducted, with adjustment for potential confounding factors. RESULTS: FIB-4 index and NFS were negatively associated with eGFR, but not UACR, after adjustment for potential confounding factors. Both FIB-4 index and NFS were significantly associated with low eGFR after adjustment for potential confounding factors. Meanwhile, in the multivariable-adjusted model, no associations were found between FIB-4 index or NFS and albuminuria. The addition of FIB-4 index or NFS to the established clinical CKD risk factors improved diagnostic accuracy of prevalence of low eGFR. We also found that there was a significant trend of higher FIB-4 index and NFS with more advanced renal fibrosis using the kidney biopsy data. CONCLUSIONS: Higher non-invasive fibrosis assessments of NAFLD were associated with higher odds of decreased eGFR.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

Mitochondrial Dysfunction in Podocytes Caused by CRIF1 Deficiency Leads to Progressive Albuminuria and Glomerular Sclerosis in Mice.

Recent studies have implicated mitochondrial disruption in podocyte dysfunction, which is a characteristic feature of primary and diabetic glomerular diseases. However, the mechanisms by which primary mitochondrial dysfunction in podocytes affects glomerular renal diseases are currently unknown. To investigate the role of mitochondrial oxidative phosphorylation (OxPhos) in podocyte dysfunction, glomerular function was examined in mice carrying a loss of function mutation of the gene encoding CR6-interacting factor-1 (CRIF1), which is essential for intramitochondrial production and the subsequent insertion of OxPhos polypeptides into the inner mitochondrial membrane. Homozygotic deficiency of CRIF1 in podocytes resulted in profound and progressive albuminuria from 3 weeks of age; the CRIF1-deficient mice also developed glomerular and tubulointerstitial lesions by 10 weeks of age. Furthermore, marked glomerular sclerosis and interstitial fibrosis were observed in homozygous CRIF1-deficient mice at 20 weeks of age. In cultured mouse podocytes, loss of CRIF1 resulted in OxPhos dysfunction and marked loss or abnormal aggregation of F-actin. These findings indicate that the OxPhos status determines the integrity of podocytes and their ability to maintain a tight barrier and control albuminuria. Analyses of the glomerular function of the podocyte-specific primary OxPhos dysfunction model mice demonstrate a link between podocyte mitochondrial dysfunction, progressive glomerular sclerosis, and tubulointerstitial diseases.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

Modulation of Renal Function in a Metabolic Syndrome Rat Model by Antioxidants in Hibiscus sabdariffa L.

Metabolic syndrome (MS) is the association of three or more pathologies among which obesity, hypertension, insulin resistance, dyslipidemia, and diabetes are included. It causes oxidative stress (OS) and renal dysfunction. Hibiscus sabdariffa L. (HSL) is a source of natural antioxidants that may control the renal damage caused by the MS. The objective of this work was to evaluate the effect of a 2% HSL infusion on renal function in a MS rat model induced by the administration of 30% sucrose in drinking water. 24 male Wistar rats were divided into 3 groups: Control rats, MS rats and MS + HSL rats. MS rats had increased body weight, systolic blood pressure, triglycerides, insulin, HOMA index, and leptin (p ≤ 0.04). Renal function was impaired by an increase in perfusion pressure in the isolated and perfused kidney, albuminuria (p ≤ 0.03), and by a decrease in clearance of creatinine (p ≤ 0.04). The activity of some antioxidant enzymes including the superoxide dismutase isoforms, peroxidases, glutathione peroxidase, glutathione-S-transferase was decreased (p ≤ 0.05). Lipoperoxidation and carbonylation were increased (p ≤ 0.001). The nitrates/nitrites ratio, total antioxidant capacity, glutathione levels and vitamin C were decreased (p ≤ 0.03). The treatment with 2% HSL reversed these alterations. The results suggest that the treatment with 2% HSL infusion protects renal function through its natural antioxidants which favor an improved renal vascular response. The infusion contributes to the increase in the glomerular filtration rate, by promoting an increase in the enzymatic and non-enzymatic antioxidant systems leading to a decrease in OS and reestablishing the normal renal function.

Cosmc-dependent mucin-type O-linked glycosylation is essential for podocyte function.

Mucin-type O-linked glycosylation, a posttranslational modification affecting the stability and biophysical characteristics of proteins, requires C1GalT1 (T synthase) and its obligate, X-linked chaperone Cosmc. Hypomorphic C1GalT1 mutations cause renal failure via not yet established mechanisms. We hypothesize that impaired Cosmc-dependent O-glycosylation in podocytes is sufficient to cause disease. Podocyte-specific Cosmc knockout mice were generated and phenotyped to test this hypothesis. Female heterozygous mice displaying mosaic inactivation of Cosmc in podocytes due to random X-linked inactivation were also examined. Mice with podocyte-specific Cosmc deletion develop profound albuminuria, foot process effacement, glomerular sclerosis, progressive renal failure, and impaired survival. Glomerular transcriptome analysis reveals early changes in cell adhesion, extracellular matrix organization, and chemokine-mediated signaling pathways, coupled with podocyte loss. Expression of the O-glycoprotein podoplanin was lost, while Tn antigen, representing immature O-glycans, was most abundantly found on podocalyxin. In contrast to hemizygous male and homozygous female animals, heterozygous female mosaic animals developed only mild albuminuria, focal foot process effacement, and nonprogressive kidney disease. Ultrastructurally, Cosmc-deficient podocytes formed Tn antigen-positive foot processes interdigitating with those of normal podocytes but not with other Cosmc-deficient cells. This suggests a cell nonautonomous mechanism for mucin-type O-glycoproteins in maintaining podocyte function. In summary, our findings demonstrated an essential and likely cell nonautonomous role for mucin-type O-glycosylation for podocyte function.