| BackgroundRenal tubulointerstitial injury in primary Sjogren’s syndrome(pSS-TIN)is the most common manifestation of renal involvement in Sjogren’s syndrome.The prevalence rate of pSS in China is as high as 0.33%to 0.77%,with 30%-50%having kidney involvement,and 75%of pSS patients undergoing renal biopsy have tubulointerstitial nephritis.Our research group found ectopic germinal centers(EGCs)in the renal tissue of pSS-TIN patients and speculated that they are closely related to the pathogenesis of pSS-TIN.B cells proliferate in the dark zone of the germinal center and receive costimulatory signals from follicular helper T cells(Tfh cells)and signals from follicular dendritic cells(FDCs)-bound antigen in the light zone of the germinal center.B cells repeatedly migrate back and forth to complete clonal expansion and antibody affinity maturation.CXCR5,a chemokine receptor,is a key condition for B cell migration.Abnormal expression of CXCR5 can lead immune cells to gather in nonlymphoid tissues(such as renal tubulointerstitium)and form EGCs.The mammalian target of rapamycin(mTOR)is a necessary condition to support the rapid proliferation and clonal expansion of B cells.Programmed death receptor 1(PD-1)is an inhibitory immune checkpoint receptor that is upregulated after T cell activation to exhaust T cells and related immune responses,maintaining normal immune tolerance;In the germinal center,PD-1 in Tfh antagonizes with inducible costimulatory molecules(ICOS),jointly controlling CXCR5 and therefore the location and activity of Tfh cells.The expression level of PD-1 in the salivary gland of patients with pSS is increased and positively correlated with EULAR Sjogren’s syndrome disease activity index(ESSDAI),indicating possible dysfunction of PD-1.Then,what is the expression level of PD-1 in pSS-TIN patients’ renal tissue?Is it related to the manifestation of ESSDAI?Given EGC is important in the pathogenesis of pSS-TIN,CXCR5 and mTOR play a key role in the formation of EGC,whether inhibiting CXCR5 and mTOR can treat pSS-TIN?There is currently no relevant research.Purpose1.Retrospective analyze the clinical and pathological characteristics and prognosis of pSSTIN patients;2.Establish a Sjogen’s Syndrome-kidney injury mouse model and evaluate their renal expression levels of PD-1,mTOR,and CXCR5;3.Inhibit renal CXCR5 and mTOR with drugs in the Sjogern’s Syndrome-kidney injury mouse model,and observe how the disease manifestations change.Methods1.A total of 136 inward patients were diagnosed as pSS-TIN in Peking Union Medical College Hospital from January 1993 to April 2023.Their demography,clinical and pathological manifestations,blood and urine tests,treatment,and prognosis were reviewed and retrospectively analyzed.The main endpoint event was defined as endstage renal disease or death.2.Take the renal cortex of C57BL/6J female mice under sterile conditions,fully digest with collagenase IV,use 70 μM and 40 μM sieve to filter away the undigested tissue and glomeruli,then obtain renal tubular tissue by centrifugation,which was resuspended with PBS and fully fragmented by centrifugation.Collect the supernatant,namely the original solution of renal tubular antigen.The concentration was tested by the BCA protein quantification method,and diluted to 2 mg/mL with sterile PBS.For the model group mice,inject 100μ L/20g body weight emulsifier prepared with renal tubular antigen and Freund’s complete adjuvant,which has a final renal tubular antigen concentration of 1mg/mL,subcutaneously at multiple points in the inguinal area of mice on day 0 and day 7.On day 14,inject 100 μL/20g body weight emulsifier prepared with renal tubular antigen and Freund’s incomplete adjuvant where renal tubular antigen has a final concentration of 0.5mg/mL using the same method.For the control group mice,inject an equal amount of complete Freund’s adjuvant or incomplete Freund’s adjuvant during the same period using the same method.Observe whether mice scratch their lips and lick their claws.At baseline and week 6/8/11 postimmunization,collect 24-hour urine volume and test urinary output of potassium,phosphorus,total protein,and creatinine,then calculate urinary phosphorus and potassium excretion scores.Collect blood serum at the baseline and week 6/8/11 postimmunization,and measure the serum creatinine,urea nitrogen,potassium,and phosphorus.Measure saliva flow rate at baseline and 6,8,and 11 weeks postimmunization.After 11 weeks of modeling,use ELISA method to measure serum anti-SSA antibody and anti-SSB antibody levels,and euthanize mice to obtain pathological information in the kidneys and submandibular glands.Extract total protein from renal tissue,and investigate the expression level of KIM-1 and PD-1 using Western blotting.3.Use rapamycin and TAK-779 to inhibit mTOR and CXCR5 in the SS-kidney injury mouse model,then evaluate changes in their water intake,saliva flow rate,blood,and urinary electrolytes level.Extract total protein from renal tissue,and observe the expression condition of CXCR5,mTOR,PD-1,and KIM-1 using Western blotting.4.Use IBM SPSS Statistics 26 to analyze statistics and GraphPad Prism 8 to present graphics.Semi-quantitative analysis of protein immunoblotting was accomplished using ImageJ.The main statistical methods include the t-test and rank sum test.Results1.Clinical and pathological characteristics of pSS-TINa.This study included a total of 136 pSS-TIN patients,mainly middle-aged women,with an average age of 40.7 ± 13.5 years old and a male-to-female ratio of 1:11.4.The main renal presentations were hypokalemia(76.1%)and renal tubular acidosis(64.0%),with an average blood creatinine of 116(53-331)μ Mol/L,eGFR 51.50(12.80-130.92)ml/min/1.73m2,and patients with eGFR<60 ml/min/1.73m2 accounted for 61.0%.The pathological manifestation of the patient is acute interstitial nephritis,without obvious glomerular lesions.b.Out of 124 patients receiving glucocorticoid treatment,more than half(72 cases)were treated with immunosuppressants or biological agents.The most common therapy was glucocorticoid in combination with cyclophosphamide(45 cases,33.1%)and mycophenolate mofetil(13 cases,9.6%).There was no significant difference in eGFR between patients who received hormone alone and those who had both hormone and cyclophosphamide at each follow-up time.2.Establishing Sjogren’s syndrome model with kidney injurya.Sjogren’s syndrome:The model group mice gradually began to scratch their lips and lick their claws from the third-week postimmunization,and had an increase in water consumption(Week 8 postimmunization,the model group 6.61±0.33 g/24h vs.control group 5.04± 0.26 g/24h，p<0.01),and a decrease in saliva secretion(Week 11 postimmunization,the model group 4.45 ± 0.64 mg/min vs.control group 9.56 ±0.39 mg/min,p<0.001).Serum anti-SSA and anti-SSB antibodies increase,and the submandibular gland manifested with lymphocyte infiltration;b.Renal presentations in Sjogren’s syndrome:Compared with the control group mice,the model group mice showed an increase in urine output at Week 8 postimmunization(1.25± 0.10ml/24h vs 0.89±0.15ml/24h,p<0.01)and 24-hour urine protein quantification(2.9±0.2 g/24h vs 1.2±0.1 g/24h,p<0.001).The excretion quantity and fraction of urine potassium and urine phosphorus were 2.78 and 2.08 times higher than those of the control group,respectively.Blood potassium(3.5±0.2mmol/L vs 4.5±0.2mmol/L,p<0.001)and blood phosphorus(1.69±0.06mmol/L vs 2.52 ±0.10mmol/L)were decreased.c.Renal pathology in Sjogren’s syndrome:Multiple focal lymphocyte aggregates were observed in the renal interstitium of the model group mice.Immunoblotting showed a significant increase in the renal expression of KIM-1,a marker of renal tubular injury,compared to the control group.There was no significant difference in the expression levels of PD-1 in renal tissue compared to the control group.3.The effect of inhibiting CXCR5 or mTOR on the Sjogren’s syndrome mice with renal injurya.Rapamycin treatment for Sjogren’s syndrome mice model:increased salivary flow rate(7.00 ± 0.45 mg/min vs 4.45± 0.64 mg/min,p<0.01),decreased water intake(4.49±0.22 g/24h vs 6.51±0.39 g/24h,p<0.01),and decreased urine output(0.55±0.10 mL/24h vs 1.31±0.09 mL/24h,p<0.001).The levels of blood uric acid(304±5 umol/L vs 238±26 umol/L,p<0.05)and blood phosphorus(2.64 ± 0.07 mmol/L vs 2.01±0.12 mmol/L,p<0.01)increased,while the amount of uric acid(0.46±0.11 mmol/24h vs 0.99±0.11 mmol/24h,p<0.05)decreased.The expression level of KIM-1,a marker of renal tubular injury in renal tissue,was significantly reduced compared to those without treatment,and the renal expression level of mTOR was reduced.b.TAK-779 treatment for Sjogren’s syndrome mice model:increased saliva flow rate(6.86 ± 0.70 mg/min vs 4.45 ± 0.64 mg/min,p<0.05),decreased water intake(3.99±0.64 g/24h vs 6.51±0.39 g/24h,p<0.01),and increased urine output(0.59±0.08 mL/24h vs 1.31±0.09 mL/24h,p<0.001).Blood phosphorus levels increased(2.39±0.11 mmol/L compared to 2.01±0.12 mmol/L,p<0.05),24-hour urine total protein quantification(1.0±0.2g/24h compared to 1.7±0.2g/24h,p<0.05),and uric acid content decreased(0.52 ±0.09mmol/24h compared to 0.99±0.11mmol/24h).The expression of renal tubular injury marker KIM-1(0.39±0.05 vs 0.83±0.08,p<0.01)was significantly reduced compared to those without treatment,while the expression of chemokine receptor CXCR5 was reduced.ConclusionsIn current study,we observed1.pSS-TIN is more common in middle-aged women,with the main manifestations of kidney damage being hypokalemia,renal tubular acidosis,and renal dysfunction.Hormone therapy alone and hormone combined with immunosuppressants can both effectively improve renal function in patients.There is no significant difference in eGFR between the two treatment regimens at each follow-up time.2.Successfully established a Sjogren’s syndrome mice with kidney injury model:the model group mice experienced scratching of the lips and licking of the claws,increased water intake,decreased saliva secretion,elevated serum anti-SSA and anti-SSB antibodies,and lymphocyte infiltration in the submandibular gland.In terms of kidney involvement,the model group mice showed an increase in urine output and 24-hour urine protein quantification,an increase in urinary potassium and phosphorus excretion,and excretion scores,while blood potassium and phosphorus decreased.Multiple focal lymphocyte aggregations were observed in the renal interstitium of the model group mice.Renal expression of KIM-1 increased.There was no significant difference in the expression of PD-1 in the renal tissue between the model group and the control group mice.3.The administration of mTOR inhibitor rapamycin and chemokine receptor CXCR5 inhibitor TAK-779 can improve the manifestations of Sjogren’s syndrome and kidney injury.After continuous use of TAK-779 treatment in the Sjogren’s syndrome model with kidney injury for 3 weeks,the expression of CXCR5 in renal tissue is inhibited,the saliva flow rate increased and water consumption decreases;The 24-hour urine total protein quantification and uric acid content decreased,blood phosphorus levels increased,and the expression of renal tubular injury marker KIM-1 was significantly reduced compared to the modeling group.Continuous use of rapamycin in the treatment of Sjogren’s syndrome model with kidney injury for 3 weeks resulted in inhibition of renal mTOR expression;The saliva flow rate of mice increased,the amount of water intake and urine output decreased,the levels of blood uric acid and phosphorus increased,and the total amount of 24-hour urine uric acid decreased;The expression level of KIM-1 decreased compared to the modeling group. |
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