The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury.

BACKGROUND: The hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive. METHODS: We tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes. RESULTS: Compared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects. CONCLUSIONS: We elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.

Sildenafil Prevents Podocyte Injury via PPAR-γ-Mediated TRPC6 Inhibition.

Transient receptor potential channel C6 (TRPC6) gain-of-function mutations and increased TRPC6 expression in podocytes induce glomerular injury and proteinuria. Sildenafil reduces TRPC6 expression and activity in nonrenal cell types, although the mechanism is unknown. Peroxisome proliferator-activated receptor γ (PPAR-γ) is a downstream target of sildenafil in the cyclic guanosine monophosphate (cGMP)-activated protein kinase G (PKG) axis. PPAR-γ agonists, like pioglitazone, appear antiproteinuric. We hypothesized that sildenafil inhibits TRPC6 expression in podocytes through PPAR-γ-dependent mechanisms, thereby counteracting podocyte injury and proteinuria. Treatment with sildenafil, the cGMP derivative 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP), or pioglitazone dose-dependently downregulated podocyte injury-induced TRPC6 expression in vitro Knockdown or application of antagonists of PKG or PPAR-γ enhanced TRPC6 expression in podocytes and counteracted effects of sildenafil and 8-Br-cGMP. We observed similar effects on TRPC6 promoter activity and TRPC6-dependent calcium influx. Chromatin immunoprecipitation showed PPAR-γ binding to the TRPC6 promoter. Sildenafil or pioglitazone treatment prevented proteinuria and the increased TRPC6 expression in rats with adriamycin-induced nephropathy and mice with hyperglycemia-induced renal injury. Rats receiving PPAR-γ antagonists displayed proteinuria and increased podocyte TRPC6 expression, as did podocyte-specific PPAR-γ knockout mice, which were more sensitive to adriamycin and not protected by sildenafil. Thus, sildenafil ameliorates podocyte injury and prevents proteinuria through cGMP- and PKG-dependent binding of PPAR-γ to the TRPC6 promoter, which inhibits TRPC6 promoter activity, expression, and activity. Because sildenafil is approved for clinical use, our results suggest that additional clinical study of its antiproteinuric effect in glomerular disease is warranted.

1,25-Vitamin D3 Deficiency Induces Albuminuria.

Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.

Vitamin D attenuates proteinuria by inhibition of heparanase expression in the podocyte.

The glomerular filtration barrier consists of podocytes, the glomerular basement membrane, and endothelial cells covered with a glycocalyx. Heparan sulphate (HS) in the glomerular filtration barrier is reduced in patients with proteinuria, which is associated with increased expression of the HS-degrading enzyme heparanase. Previously, we showed that heparanase is essential for the development of proteinuria in experimental diabetic nephropathy. Vitamin D supplementation reduces podocyte loss and proteinuria in vitro and in vivo. Therefore, we hypothesize that vitamin D reduces proteinuria by reducing glomerular heparanase. Adriamycin-exposed rats developed proteinuria and showed increased heparanase expression, which was reduced by 1,25-dihydroxyvitamin D3 (1,25-D3) treatment. In vitro, adriamycin increased heparanase mRNA in the podocyte, which could be corrected by 1,25-D3 treatment. In addition, 1,25-D3 treatment reduced transendothelial albumin passage after adriamycin stimulation. In line with these results, we showed direct binding of the vitamin D receptor to the heparanase promoter, and 1,25-D3 dose-dependently reduced heparanase promoter activity. Finally, 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice developed proteinuria and showed increased heparanase, which was normalized by 1,25-D3 treatment. Our data suggest that the protective effect of vitamin D on the development of proteinuria is mediated by inhibiting heparanase expression in the podocyte.

Glucose specifically regulates TRPC6 expression in the podocyte in an AngII-dependent manner.

Slit diaphragm and podocyte damage is crucial in the pathogenesis of proteinuria in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and inducing podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP.

Vitamin D down-regulates TRPC6 expression in podocyte injury and proteinuric glomerular disease.

The transient receptor potential cation channel C6 (TRPC6) is a slit diaphragm protein expressed by podocytes. TRPC6 gain-of-function mutations cause autosomal dominant focal segmental glomerulosclerosis. In acquired proteinuric renal disease, glomerular TRPC6 expression is increased. We previously demonstrated that acquired increased TRPC6 expression is ameliorated by antiproteinuric angiotensin receptor blockers and angiotensin-converting enzyme inhibitors. Vitamin D also has an antiproteinuric effect. We hypothesized that vitamin D reduces proteinuria by affecting TRPC6 expression in podocytes. Adriamycin-induced nephropathy increased TRPC6 mRNA and protein expression and induced proteinuria in rats. Treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) normalized TRPC6 expression and reduced proteinuria. In vitro, podocyte injury induced by adriamycin exposure in cultured podocytes increased TRPC6 expression. Treatment of injured podocytes with 1,25-D3 dose dependently reduced adriamycin-induced TRPC6 expression. Chromatin immunoprecipitation analysis demonstrated that the vitamin D receptor directly binds to the TRPC6 promoter. Moreover, 1,25-D3 reduced TRPC6 promoter activity in a luciferase reporter assay. In 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice, TRPC6 expression was increased, accompanied by podocyte foot process effacement and proteinuria. 1,25-D3 supplementation normalized TRPC6 expression, podocyte morphology, and proteinuria in these mice. These results demonstrate that vitamin D down-regulates the enhanced TRPC6 expression in in vivo and in vitro podocyte injury, possibly through a direct effect on TRPC6 promoter activity. This TRPC6 down-regulation could contribute to the antiproteinuric effect of vitamin D.

Peroxisome proliferator-activated receptor ɣ agonist mediated inhibition of heparanase expression reduces proteinuria.

BACKGROUND: Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor É£ (PPARÉ£) agonists can ameliorate proteinuria. Since a recent study showed that PPARÉ£ regulates HPSE expression in liver cancer cells, we hypothesized that PPARÉ£ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. METHODS: Regulation of HPSE by PPARÉ£ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARÉ£ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARÉ£ agonist pioglitazone. FINDINGS: Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARÉ£ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARÉ£ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. INTERPRETATION: PPARÉ£-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. FUNDING: This study was financially supported by the Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships).

IL-1ß Promotes a New Function of DNase I as a Transcription Factor for the Fas Receptor Gene.

Recently we described that endonuclease inactive DNase I translocated into the nucleus in response to increased endogenous IL-1ß expression. Here, we demonstrate impact and function of translocated DNase I in tubular cells. Effect of cytokines on expression level and nuclear localisation of DNase I and corresponding levels of Fas receptor (FasR) and IL-1ß were determined by confocal microscopy, qPCR and western blot analyses, in presence or absence of siRNA against IL-1ß and DNase I mRNA. Nuclear DNase I bound to the FAS promotor region as determined by chromatin immuno-precipitation analysis. Data demonstrate that; (i) translocation of DNase I depended on endogenous de novo-expressed IL-1ß, (ii) nuclear DNase I bound FAS DNA, (iii) FasR expression increased after translocation of DNase I, (iv) interaction of exogenous Fas ligand (FasL) with upregulated FasR induced apoptosis in human tubular cells stimulated with TNFα. Thus, translocated DNase I most probably binds the promoter region of the FAS gene and function as a transcription factor for FasR. In conclusion, DNase I not only executes chromatin degradation during apoptosis and necrosis, but also primes the cells for apoptosis by enhancing FasR expression.

Early development of hyperparathyroidism due to loss of PTH transcriptional repression in patients with HNF1ß mutations?

CONTEXT: Heterozygous mutations or deletions of the transcription factor hepatocyte nuclear factor 1ß (HNF1ß) result in a heterogeneous syndrome characterized by renal cysts and diabetes, together with a variety of other extrarenal and renal manifestations. Interestingly, in several patients with HNF1ß abnormalities, we observed early hyperparathyroidism and PTH levels that we judged inappropriately high compared with the degree of renal function decline. OBJECTIVE: Based on the above clinical observations, we tested the hypothesis of a direct role of HNF1ß in the transcriptional regulation of the human PTH gene in the parathyroid gland. DESIGN, SETTING, AND PATIENTS: Immunostaining of human parathyroid sections, RT-PCR, chromatin immunoprecipitation (ChIP), and luciferase reporter assays in human embryonic kidney cells (HEK293) were performed. We eventually report clinical data from all 11 HNF1ß patients known at our institute, 9 with heterozygous HNF1ß whole-gene deletions and 2 with heterozygous HNF1ß mutations. RESULTS: PTH levels were high in 8 patients. In 2 of these patients, the hyperparathyroidism was clearly appropriate for the level of kidney function, whereas PTH might be discrepant in the others. We demonstrated HNF1ß expression in PTH-positive cells of human parathyroid gland. Chromatin immunoprecipitation analysis showed that HNF1ß directly binds responsive elements within the human PTH promoter. Cotransfection of a PTH promoter- luciferase construct with a wild-type HNF1ß construct resulted in a maximal reduction of 30% of PTH promoter activity. Importantly, HNF1ß mutants lacked this inhibitory property. Serial deletions in the PTH promoter construct revealed that the inhibitory effect of HNF1ß resides between -200 and -70 bp from the transcription initiation site. CONCLUSIONS: Our data demonstrate that HNF1ß is a novel repressor of human PTH gene transcription, which could contribute to the development of hyperparathyroidism in patients with HNF1ß mutations or deletions.